Abstract
HER2+ breast leptomeningeal carcinomatosis (HER2+ LC) occurs when tumor cells spread to cerebrospinal fluid–containing leptomeninges surrounding the brain and spinal cord, a complication with a dire prognosis. HER2+ LC remains incurable, with few treatment options. Currently, much effort is devoted toward development of therapies that target mutations. However, targeting epigenetic or transcriptional states of HER2+ LC tumors might efficiently target HER2+ LC growth via inhibition of oncogenic signaling; this approach remains promising but is less explored. To test this possibility, we established primary HER2+ LC (Lepto) cell lines from nodular HER2+ LC tissues. These lines are phenotypically CD326+CD49f−, confirming that they are derived from HER2+ LC tumors, and express surface CD44+CD24−, a cancer stem cell (CSC) phenotype. Like CSCs, Lepto lines showed greater drug resistance and more aggressive behavior compared with other HER2+ breast cancer lines in vitro and in vivo. Interestingly, the three Lepto lines overexpressed Jumonji domain–containing histone lysine demethylases KDM4A/4C. Treatment with JIB04, a selective inhibitor of Jumonji demethylases, or genetic loss of function of KDM4A/4C induced apoptosis and cell-cycle arrest and reduced Lepto cell viability, tumorsphere formation, regrowth, and invasion in vitro. JIB04 treatment of patient-derived xenograft mouse models in vivo reduced HER2+ LC tumor growth and prolonged animal survival. Mechanistically, KDM4A/4C inhibition downregulated GMCSF expression and prevented GMCSF-dependent Lepto cell proliferation. Collectively, these results establish KDM4A/4C as a viable therapeutic target in HER2+ LC and spotlight the benefits of targeting the tumorigenic transcriptional network.
HER2+ LC tumors overexpress KDM4A/4C and are sensitive to the Jumonji demethylase inhibitor JIB04, which reduces the viability of primary HER2+ LC cells and increases survival in mouse models.
Introduction
HER2+ breast leptomeningeal carcinomatosis (HER2+ LC) occurs when tumor cells spread from the breast to the cerebrospinal fluid (CSF)-containing leptomeninges surrounding the brain and spinal cord (1, 2). HER2+ LC is a rare but devastating “late-stage” complication of systemic cancers, and once diagnosed patients usually have a median survival of approximately 15 weeks (3). Among HER2+ LC cases, 30% are diagnosed after a substantial disease-free interval, although the condition can also occur as the first manifestation of cancer (4–6). Given that cancer subtypes differ, HER2+ LCs from breast cancer possess different natural histories than leptomeningeal carcinomatosis derived from other cancers and likely will respond to treatment differently (7). As compared with other solid tumors, survival varies by etiology, with patients with breast cancer having the best prognosis (13%–25% survival at 1 year and 6% at 2 years; ref. 8). Although there has been significant progress in treating breast cancer by targeting systemic disease, efficacy of those treatments in the central nervous system remains a significant challenge, with HER2+ LC typically developing while the systemic tumor burden is well managed (9–11). Despite advances in targeted radiation and chemotherapy, survival remains poor after HER2+ LC diagnosis, averaging 3–6 months (1). Management of HER2+ LC currently requires a multidisciplinary approach with radiotherapy and intrathecal therapy. Unfortunately, response rates to current treatments are often <20% (12, 13).
Several studies report dysregulation of both transcription factors and epigenetic regulators in various solid tumors (14–17). Relevant to the latter, various groups have focused on developing cofactor analogs to modulate activities of epigenetic enzymes overexpressed in tumors (15). These studies have identified several promising cell-permeable small molecule inhibitors of histone-modifying enzymes, such as DOT1Li, EPZ004777, LSD1i, and GSK690 (18–22). However, some cell-permeable inhibitors with high potency in vitro have shown little or no biological activity in vivo (23, 24).
Here, we generated the first-ever expandable primary HER2+ LC cell lines and patient-derived xenograft (PDX) models and used them to identify new epigenetic vulnerabilities present specifically in HER2+ LC tumor cells. Specifically, we screened a library of 181 small molecules targeting epigenetic factors to identify inhibitors HER2+ LC tumorsphere viability at sub-μmol/L dosage. The most promising candidate was the cell-permeable small molecule JIB04, which inhibits Jumonji domain histone demethylases in vitro. We then identified the lysine demethylases KDM4A/4C as novel targets in HER2+ LC. Separately, both JIB04-mediated inhibition and (RNAi)-based knockdown of KDM4A/4C in Lepto cells induced apoptosis and cell-cycle arrest and reduced cell viability, tumorsphere formation, regrowth, and invasion in vitro. In vivo, JIB04 treatment reduced HER2+ LC tumor growth in PDX NOD/SCID mouse models and prolonged survival. Mechanistically, we showed that KDM4A/4C knockdown downregulates GMCSF and GMCSF-dependent proliferation of Lepto cells via autocrine signaling through GMCSFRα. Overall, we have identified a novel small molecule inhibitor of Jumonji enzymes with selective anticancer properties against HER2+ LC–derived Lepto cell lines and HER2+ LC tumors in vitro and in vivo. Thus, our work provides the necessary impetus to develop targeted inhibitors of KDM4A/KDM4C for use in HER2+ LC tumors.
Materials and Methods
Ethics statements
The use of human specimens was approved by the City of Hope (COH; Duarte, CA) Institutional Review Board (IRB; protocols #07047 and #16015; refs. 25–27). Written informed consent was obtained from all the patients under protocol #07047, and the study was conducted in accordance with the Declaration of Helsinki, institutional guidelines, and all local, state, and federal regulations. All mouse studies were approved by the COH (Duarte, CA) Institutional Animal Care and Use Committee (IACUC; protocol #10044). Because HER2+ leptomeningeal carcinomatosis occurs predominantly in females, we used only female NOD/SCID mice for all in vivo experiments.
Reagents
List of all reagents (compounds/drugs, antibodies, culture media, media supplements, and other reagents) and software used, along with their source information and research resource identifier numbers, are included in the Supplementary Tables S1–S3.
Derivation and culture of Lepto cell lines
Fresh nodular HER2+ LC specimens were acquired from consented patients who underwent surgeries to acquire meningeal biopsies for pathologic confirmation of HER2+ LC or to decompress localized symptomatic lesions (IRB protocols #07047 and 16015). A portion of each specimen was mechanically and/or enzymatically dissociated, and EpCAM+ (CD326+) CD49f− and CD44+CD24− cells [representing epithelial and cancer stem cell (CSC) phenotypes] were sorted by FACS and maintained in an incubator at 37°C and 5% CO2. Two primary cell lines, Lepto1 and Lepto2, were derived from two patients. Cryobanks of low-passage Lepto1 and Lepto2 cells were created using Stemcell banker cryopreservation media (AMSBIO) and banked in liquid nitrogen at −180°C. The isolated primary cells were cultured in human CSF (hCSF) and/or Advanced DMEM/F-12 (Dulbecco's Modified Eagle Medium/Ham's F-12 Nutrient Mixture; Life Technologies) with various supplements (Supplementary Table S2) on collagen-coated T-75 flasks, as described previously (26). All cell lines were used between passages 10 and 35. Cell lines were tested for Mycoplasma contamination via PCR (Agilent Mycosenser Mycoplasma Assay Kit) as recent as 2 months prior to last experiments.
Cell lines
Cell lines (HEK293T, MDA-MB-231, BT-474, MCF7, MCF10A, T47D, and SK-BR3) were obtained from ATCC (details listed in Supplementary Table S1). All the lines were monthly monitored for Mycoplasma contamination. All the cell lines were either grown in RPMI + 10% FBS + penicillin-streptomycin (1×), IMDM +10% FBS + penicillin-streptomycin (1×) or RPMI + 20% FBS + penicillin-streptomycin (1×) according to guidance from ATCC. HEK293T were cultured in DMEM + 10% FBS + Penn-Strep (1×). Cell line authentication was performed by short tandem repeat profiling at the IDEXX Bioanalytic Laboratories Inc and tested as Mycoplasma negative by PCR (Agilent Mycosenser Mycoplasma Assay Kit) as recent as 1 month prior to last experiments.
Mouse studies and in vivo drug administration
For PDX model development, only 4–6 weeks old female NOD/SCID mice were used, which were maintained under specific pathogen-free conditions in accordance with guidelines and therapeutic interventions approved by COH IACUC. Because HER2+ leptomeningeal carcinomatosis occurs predominantly in females, we used only female NOD/SCID mice for all in vivo experiments. For the survival study, NOD/SCID 6–8 weeks old female mice were utilized. Animals were housed under standard conditions in the ARCH facility at COH (Duarte, CA). All animal experiments were carried out under approved IACUC protocols and followed COH's animal care procedures.
In vivo PDX models
To evaluate tumor growth and survival, mCherry and firefly luciferase (mCherry: LUC, Addgene_75020) transduced Lepto1 and/or Lepto2 cell lines were injected via cisterna magna puncture (200 K cells in 20 μL PBS) into cohorts of 6–8 weeks old, female NOD/SCID mice (n = 12/group). Following randomization of animals into control and experimental groups at day 7, post-implantation of Lepto cells, the mice were intraperitoneally injected with JIB04 (60 or 110 mg/kg; experimental group) in PBS or vehicle or PBS alone (control group). Tumor growth was monitored weekly using bioluminescence imaging (BLI) on a Xenogen Imaging System (Xenogen Corp) in a double-blinded fashion. At the conclusion of experiments, mice were euthanized, brains and tumors were harvested and fixed and subjected to Western blot analysis.
IHC and immunofluorescence staining
Mouse brains were fixed in 4% paraformaldehyde, dehydrated in 30% sucrose, and then sectioned (10 μm) and mounted on microscopic slides. Immunofluorescence (IF) staining with GFAP, HER2, and MBP antibody and hematoxylin and eosin staining for tumor growth were done as described previously (25, 26).
Statistical analyses
Data presented in the figures are mean values ± SE, using data generated from n = 3 biological replicates with n = 2 technical replicates present in each biological replicate. Statistical significance between groups was determined using one- or two-way ANOVA, followed by multiple comparisons with Bonferroni multiple comparisons correction. The level of significance used was α:0.05. Other statistical evaluations were performed using the Student t test. The software used for the above-mentioned analyses was GraphPad Prism 8.4.1. Kaplan–Meier curve was used to model overall survival. A P value of <0.05 was considered statistically significant. Significance in statistical analyses in the figures are *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
All additional methods are described in the Supplementary Material and Methods section.
Results
Derivation and characterization of Lepto cell lines from various nodular HER2+ LC tumor tissues
Development of effective treatment for HER2+ LC requires establishment HER2+ LC models that recapitulate human disease (28). To meet this need, we derived three primary Lepto lines (Lepto1, Lepto2, and Lepto3) from fresh nodular tumor tissues obtained from 3 patients with pathological confirmation of HER2+ LC. Briefly, tissues were enzymatically dissociated into single-cell suspensions and FACS sorted to enrich for cells exhibiting a breast epithelial CSCs phenotype (CD326+CD49f− and CD44+CD24−). Cells were expanded and cultured on collagen-coated plates in hCSF-supplemented media, and then production was scaled up to produce a master cryobank batch of low-passage primary Lepto cells used in experiments reported below (see schematic in Fig. 1A).
Derivation and characterization of Lepto cell lines from various nodular HER2+ LC tumor tissues. A, Scheme illustrating the collection of human tumor samples, derivation, and maintenance of Lepto1, Lepto2, and Lepto3 cell lines on collagen-coated plates, scale-up of low-passage cell lines followed by in vivo functional characterization, and unbiased tumorsphere viability screening via usage of drugs that target various epigenetic factors/genes. B, BLI-based quantification of brain-tropic breast cancer MDA-MB-231 (231-BR), BBM cells, and HER2+ LC (Lepto1) cells in the brain and spinal cord of xenografted NOD/SCID mouse models. ***, P < 0.001. C, Representative BLI imaging of mice xenografted with 231-BR, BBM, and Lepto1 cells in the brain and spinal cord of PDX models. D, Kaplan–Meier survival analyses in days post-implantation showing shorter survival of mice bearing BBM and Lepto1 compared with mice bearing 231-BR cells. E, Top, histopathologic analysis of the (hematoxylin and eosin stained) sagittal section of the brain and axial spinal cord showing Lepto1 deposition in the CNS with invasion of the brain and spinal cord parenchyma. Bottom, Hematoxylin and eosin staining at higher magnification showing the presence of dark stained Lepto1 cells. F, Top, IF images of whole brain sections collected from mice 30 days after Lepto1 cell implantation. Imaging of OPCs (Olig2, purple), astrocytes (GFAP, green). Bottom, Lepto1 cells (mCherry, red) shows a layer of reactive astrocytes on the surface of the brain stem, juxtaposed to nodular HER2+ LC. G, FACS-based analyses showing that approximately 95% of Lepto1 cells are CD326+CD49f−, with most of those also CD44+CD24−, indicative of a CSC phenotype. H, Quantification of tumorspheres formed from 100 Lepto1, Lepto2, Lepto3, BT-474, and SK-BR3 cells. Means ± SD of three technical and three biological replicates are shown. ***, P < 0.0001. I, Representative images of tumorspheres generated from Lepto1, Lepto2, Lepto3, BT-474, and SK-BR3 cells. Scale bar, 100 μm. J, Dose–response curves of Lepto1, BT-474, and SK-BR3 cells treated 48 hours with methotrexate. Means ± SD of three technical replicates and three biological replicates are shown. K, Viability of Lepto1, Lepto2, Lepto3, BT-474, and SK-BR3 cells treated 48 hours with various concentrations of lapatinib, trastuzumab, and cytarabine. Means ± SD of three technical replicates and three biological replicates are shown. *, P < 0.001.
Derivation and characterization of Lepto cell lines from various nodular HER2+ LC tumor tissues. A, Scheme illustrating the collection of human tumor samples, derivation, and maintenance of Lepto1, Lepto2, and Lepto3 cell lines on collagen-coated plates, scale-up of low-passage cell lines followed by in vivo functional characterization, and unbiased tumorsphere viability screening via usage of drugs that target various epigenetic factors/genes. B, BLI-based quantification of brain-tropic breast cancer MDA-MB-231 (231-BR), BBM cells, and HER2+ LC (Lepto1) cells in the brain and spinal cord of xenografted NOD/SCID mouse models. ***, P < 0.001. C, Representative BLI imaging of mice xenografted with 231-BR, BBM, and Lepto1 cells in the brain and spinal cord of PDX models. D, Kaplan–Meier survival analyses in days post-implantation showing shorter survival of mice bearing BBM and Lepto1 compared with mice bearing 231-BR cells. E, Top, histopathologic analysis of the (hematoxylin and eosin stained) sagittal section of the brain and axial spinal cord showing Lepto1 deposition in the CNS with invasion of the brain and spinal cord parenchyma. Bottom, Hematoxylin and eosin staining at higher magnification showing the presence of dark stained Lepto1 cells. F, Top, IF images of whole brain sections collected from mice 30 days after Lepto1 cell implantation. Imaging of OPCs (Olig2, purple), astrocytes (GFAP, green). Bottom, Lepto1 cells (mCherry, red) shows a layer of reactive astrocytes on the surface of the brain stem, juxtaposed to nodular HER2+ LC. G, FACS-based analyses showing that approximately 95% of Lepto1 cells are CD326+CD49f−, with most of those also CD44+CD24−, indicative of a CSC phenotype. H, Quantification of tumorspheres formed from 100 Lepto1, Lepto2, Lepto3, BT-474, and SK-BR3 cells. Means ± SD of three technical and three biological replicates are shown. ***, P < 0.0001. I, Representative images of tumorspheres generated from Lepto1, Lepto2, Lepto3, BT-474, and SK-BR3 cells. Scale bar, 100 μm. J, Dose–response curves of Lepto1, BT-474, and SK-BR3 cells treated 48 hours with methotrexate. Means ± SD of three technical replicates and three biological replicates are shown. K, Viability of Lepto1, Lepto2, Lepto3, BT-474, and SK-BR3 cells treated 48 hours with various concentrations of lapatinib, trastuzumab, and cytarabine. Means ± SD of three technical replicates and three biological replicates are shown. *, P < 0.001.
To compare metastatic organotropism, we first transduced the Lepto1, as well as breast to brain metastatic tumor lines (BBM; ref. 29) and the brain-tropic breast cancer line MDA-MB-231 (231-BR cells) with lentiviruses encoding mCherry and firefly luciferase (mCherry:LUC). We then injected cells (100 K from each line) into the CSF space via the cisterna magna puncture of NOD/SCID mice (n = 4) and monitored tumor cell growth over time (∼28 days) by BLI (Fig. 1B and C). In addition, although we observed fewer metastatic Lepto1 cells than 231-BR and BBM cells in brain, overall, there was a greater number of metastatic Lepto1 cells across both brain and spinal cord (Fig. 1B and C). Furthermore, Kaplan–Meier curve analysis showed that NOD/SCID mice harboring Lepto1 cells had significantly shorter survival periods than mice bearing BBM and 231-BR cells (Fig. 1D). As expected, BBM and 231-BR cells generated tumors only within the brain parenchyma (29). In contrast, Lepto1 cell bioluminescence appeared on the surface as well as in the parenchyma of the brain and spinal cord (Fig. 1E). Histologic examination confirmed that Lepto1 cells colonized the leptomeningeal surface of the brain, brain stem, and spinal cord (Fig. 1E). When we performed IF staining of brain tissue sections harvested at euthanasia on day 30, staining of Lepto1 using mCherry and glial cell populations (with anti-GFAP antibodies) indicated astrocytes juxtaposed to HER2+ LC tumors (Fig. 1F; Supplementary Fig. S1A–S1C). In contrast, the inner core of the tissue, which is rich in Olig2-positive oligodendrocyte progenitor cells (OPC), lacked Lepto cells [Fig. 1F (Lepto1); Supplementary Fig. S1C (Lepto2 and 3)]. BLI quantification on day 16 indicated the presence of Lepto1-derived tumors on the surface of the brain and spinal cord, while BBM and 231-BR bearing NOD/SCID mice did not show spinal cord metastasis (Fig. 1B–E), and that there were a greater number of metastatic Lepto1 cells in spinal cord than in brain, relative to BBM and 231-BR–derived tumors (Fig. 1B–E). This observation is consistent with known distribution of HER2+ LC tumors in patients, in which white matter is relatively HER2+ LC tumor resistant (30), while tumors develop primarily in leptomeningeal regions of spinal cord and brain.
FACS analysis demonstrated that Lepto lines showed homogeneous surface expression of CD326+ and lacked surface expression of CD49f [Fig. 1G; Supplementary Fig. S2A (top)], consistent with a HER2+ LC origin. HER2+ LC tumor tissue cells had been FACS sorted for the CD326+CD49f−CD44+CD24− phenotype, and accordingly, approximately 99% of the HER2+CD326+CD49f− Lepto1 cells were CD44+CD24− [Fig. 1G; Supplementary Fig. S2A (bottom)], a phenotype associated with human breast CSCs. A property of CSCs is the ability to initiate and drive tumorigenesis, activities associated with tumor relapse and drug resistance. Next, we used Western blot analyses to confirm that Lepto1–3 lines were HER2+ (Supplementary Fig. S2B). To assess CSC-like activity in vitro, we compared tumorsphere-forming ability of Lepto1, 2, and 3 cells with that of the HER2+ breast cancer lines BT-474 and SK-BR3. Lepto1–3 cells showed significantly greater tumorsphere-forming ability (Fig. 1H and I) and significantly more rapid proliferation (Supplementary Fig. S1D) than did BT-474 or SK-BR3 cells. Furthermore, relative to SK-BR3 and BT-474 cells, fewer Lepto1–3 cells were required for tumor seeding in vivo (Supplementary Fig. S1E). Next, we assessed resistance of Lepto cells to various chemotherapeutic drugs. Relative to BT-474 or SK-BR3 cells, Lepto1–3 lines were more resistant to methotrexate, a commonly used intrathecal chemotherapy drug, with an approximately 5-fold greater IC50 (Fig. 1J). Lepto lines also exhibited 10-fold to 20-fold greater resistance to lapatinib, trastuzumab, and cytarabine relative to BT-474 or SK-BR3 cells (Fig. 1K). Thus, HER2+CD326+CD49f− Lepto cells not only model nodular HER2+ LC tissues phenotypically but demonstrate a predominant CSC (CD44+CD24−) phenotype and properties of bona fide CSCs, namely, increased drug resistance, aggressiveness, and robust capacity to form tumorspheres.
Finally, we assessed the metastatic efficiency and brain/spinal cord colonization of Lepto lines by injecting all three Lepto lines (Lepto1–3 transduced with firefly and RFP; Lepto1–3-FF-RFP) via intracardiac delivery. Following injection on day 0, all three lines established systemic metastasis, including within the brain and spinal cord by day 15 (Supplementary Fig. S3A–S3C). Lepto cells not only formed metastases in various organs but also produced significant brain [Supplementary Fig. S3C i–ii (Lepto2 and 3)] and spinal cord metastases [Supplementary Fig. S3C iii–iv (Lepto2 and 3)] in NOD/SCID mice to a comparable extent, suggesting that Lepto lines can form metastatic colonies in the leptomeningeal microenvironment of brain and spinal cord.
JIB04 effectively and specifically inhibits viability, invasiveness, tumorsphere formation, and growth of HER2+ Lepto lines and enhances their apoptosis
Given their association with relapse and metastases, CSCs are key targets in efforts to eradicate tumor cells (31). Moreover, numerous reports indicate that dysregulation of epigenetic pathways plays a role in tumorigenesis (14–17). Thus, we evaluated the epigenome of HER2+ LC tumors to identify targetable vulnerabilities. To do so, we used tumorspheres derived from Lepto1 cells to screen a library of 181 small molecule drugs that target epigenetic factors for compounds that inhibit primary tumorsphere formation or decrease tumorsphere viability (Supplementary Fig. S5A; Supplementary Table S4). Interestingly, we identified JIB04 (a pan-Jumonji domain inhibitor), trichostatin-A [a histone deacetylase (HDAC) inhibitor], chaetocin [which inhibits histone lysine methyltransferase SU(VAR)3–9], and Barasertib and AZD2461 (pan-Aurora kinase inhibitors) as effectively inhibiting tumorsphere-forming ability of primary Lepto1 lines (Supplementary Fig. S5A; Supplementary Table S4), and define them as “primary screen hits.” In agreement, based on CellTiter-Glo assays, JIB04 exposure for 4 days reduced viability of cultured Lepto1 cells at concentrations above 25 nmol/L (Fig. 2A). Moreover, JIB04 treatment of Lepto lines 1–3 reduced cell viability with more rapid kinetics than that seen in HER2+ or HER2− lines (specifically within 24 hours) and showed dose and time-dependent effects (Fig. 2A and B). Following treatment of Lepto lines 1–3 with varying JIB04 concentrations for 24 hours, cell viability decreased significantly and dose-dependently, showing a 50% reduction in viability at concentrations ranging from 0.06 to 0.2 μmol/L (Fig. 2B). Furthermore, when compared with other non-HER2+ LC–derived lines such as HER2+ (SK-BR3 and BT-474) or HER2− (MCF7, MDA-MB-231, T47D, and MCF10) breast cancer lines, JIB04 specifically targeted HER2+ LC–derived Lepto lines at significantly lower concentrations (Fig. 2B). Analysis of representative IF images of Lepto1–3 cells stained with Calcein AM Red and DAPI, markers of living cells, confirmed significant reduction in Lepto1–3 cell viability after 48 hours of exposure to 0.25 μmol/L JIB04 (Fig. 2C). To avoid potential confounding effects of prolonged JIB04 toxicity, on Lepto lines we assessed invasiveness potential and tumorsphere formation capability in cell lines pretreated with JIB04 prior to analyses. To assess invasivity, we pretreated GFP-transduced Lepto1, Lepto2, Lepto3, BT-474, and SK-BR-3 cell lines 24 hours with JIB04 (0.5 μmol/L) or DMSO and then analyzed migration using matrigel invasion chambers. Relative to DMSO pretreated controls, JIB04 pretreatment significantly inhibited invasiveness (defined as migration of GFP+ cells from the top to bottom chamber, which contained astrocyte conditioned medium) of Lepto1–3 lines but not of BT-474 or SK-BR-3 cells [Fig. 2D (representative images of wells containing Lepto1 pretreated with JIB04 or DMSO) and F].
JIB04 effectively and specifically inhibits viability, invasiveness, tumorsphere formation, and growth of HER2+ Lepto lines and enhances their apoptosis. A, CellTiter-Glo–based viability analyses demonstrated JIB04-mediated reduction in the Lepto1 cell viability in a dose- and time-dependent manner. Note a significant decrease in the Lepto1 cell viability at concentrations of 50 nmol/L and above in a time-dependent manner. Means ± SD of three technical replicates and three biological replicates are shown. B, CellTiter-Glo–based viability analyses demonstrated JIB04 reduces the viability of Lepto1, Lepto2, and Lepto3 cells, but not other HER2+ and HER2− breast cancer cells, in a dose-dependent manner. Means ± SD of four technical replicates and three biological replicates are shown. Bottom, IC50 values of Lepto1, Lepto2, Lepto3, MCF7, MCF10, SK-BR-3, BT-474, MDA-MB-231, T47D for JIB04 (μmol/L). C, Representative confocal images of Lepto1, Lepto2, and Lepto3 cells at ×10 magnification, stained with Calcein AM-Red and DAPI (for living cells) at 48 hours after start of treatment with JIB04 (0.25 μmol/L). Control cells were treated with DMSO alone. Scale bar, 50 μmol/L. D, Representative confocal images showing GFP-labeled Lepto1, BT-474, and SK-BR3 cells in Matrigel invasion chambers. Whole well image from a 6-well plate is shown. Lepto1 cells were pretreated with JIB04 (0.5 μmol/L) for 24 hours and allowed to migrate into the bottom chamber containing astrocyte conditioned medium. JIB04 pretreatment was sufficient to reduce the invasive potential of Lepto1 cells more than that of BT-474 and SK-BR-3 cell. E, Analysis of Annexin-V positivity in Lepto1, Lepto2, Lepto3, BT-474, and SK-BR-3 lines treated 48 hours with 0.5 μmol/L JIB04. Quantification of flow cytometry data is shown. Means ± SD of three technical replicates and three biological replicates are shown. *, P < 0.01; **, P < 0.001. F, Quantification of the GFP signals of Lepto1, Lepto2, Lepto3, BT-474, and SK-BR-3 cells treated as shown in D. Means ± SD of three technical replicates and three biological replicates are shown. ***, P < 0.001. G, Representative FACS plots showing Annexin-V levels in CD326+ Lepto1 cells incubated with JIB04 (0.25 μmol/L) for 24, 48, and 72 hours. H, Chemical structure of JIB04.
JIB04 effectively and specifically inhibits viability, invasiveness, tumorsphere formation, and growth of HER2+ Lepto lines and enhances their apoptosis. A, CellTiter-Glo–based viability analyses demonstrated JIB04-mediated reduction in the Lepto1 cell viability in a dose- and time-dependent manner. Note a significant decrease in the Lepto1 cell viability at concentrations of 50 nmol/L and above in a time-dependent manner. Means ± SD of three technical replicates and three biological replicates are shown. B, CellTiter-Glo–based viability analyses demonstrated JIB04 reduces the viability of Lepto1, Lepto2, and Lepto3 cells, but not other HER2+ and HER2− breast cancer cells, in a dose-dependent manner. Means ± SD of four technical replicates and three biological replicates are shown. Bottom, IC50 values of Lepto1, Lepto2, Lepto3, MCF7, MCF10, SK-BR-3, BT-474, MDA-MB-231, T47D for JIB04 (μmol/L). C, Representative confocal images of Lepto1, Lepto2, and Lepto3 cells at ×10 magnification, stained with Calcein AM-Red and DAPI (for living cells) at 48 hours after start of treatment with JIB04 (0.25 μmol/L). Control cells were treated with DMSO alone. Scale bar, 50 μmol/L. D, Representative confocal images showing GFP-labeled Lepto1, BT-474, and SK-BR3 cells in Matrigel invasion chambers. Whole well image from a 6-well plate is shown. Lepto1 cells were pretreated with JIB04 (0.5 μmol/L) for 24 hours and allowed to migrate into the bottom chamber containing astrocyte conditioned medium. JIB04 pretreatment was sufficient to reduce the invasive potential of Lepto1 cells more than that of BT-474 and SK-BR-3 cell. E, Analysis of Annexin-V positivity in Lepto1, Lepto2, Lepto3, BT-474, and SK-BR-3 lines treated 48 hours with 0.5 μmol/L JIB04. Quantification of flow cytometry data is shown. Means ± SD of three technical replicates and three biological replicates are shown. *, P < 0.01; **, P < 0.001. F, Quantification of the GFP signals of Lepto1, Lepto2, Lepto3, BT-474, and SK-BR-3 cells treated as shown in D. Means ± SD of three technical replicates and three biological replicates are shown. ***, P < 0.001. G, Representative FACS plots showing Annexin-V levels in CD326+ Lepto1 cells incubated with JIB04 (0.25 μmol/L) for 24, 48, and 72 hours. H, Chemical structure of JIB04.
Next, we analyzed apoptosis based on Annexin-V staining in Lepto1, Lepto2, Lepto3 BT-474, and SK-BR3 cells after JIB04 treatment (0.5 μmol/L) for 48 hours. JIB04 induced significantly higher levels of apoptosis in Lepto1–3 (∼90%) relative to BT-474 (∼20%) and SK-BR3 (∼60%) cells (Fig. 2E). Moreover, exposure of Lepto1 cells to lower (0.25 μmol/L) JIB04 concentrations from 24 to 72 hours was sufficient to induce significant apoptosis in CD326+ Lepto1 cells relative to DMSO controls (Fig. 2G). Furthermore, apoptosis of CD326+ Lepto1–3 cells, as evaluated by FACS-based analyses of Annexin V–positive cells, increased from approximately 40%, upon exposure to JIB04 for 24 hours, to approximately 90% after 72 hours of exposure [Fig. 2G; Supplementary Fig. S4A (Lepto1); Supplementary Fig. S4B (Lepto2 and 3)]. Furthermore, propidium iodide–based analyses demonstrated that JIB04 treatment induced cell-cycle arrest in Lepto1 cells relative to DMSO controls [Supplementary Fig. S4C (top and bottom panel)]. Moreover, relative to DMSO controls, JIB04 treatment of Lepto1 cells significantly decreased the number of cells in G1-phase of the cell cycle and increased the number in S and G2–M [Supplementary Fig. S4C (top and bottom)]. Structure of JIB04 is shown in Fig. 2H.
CSCs have an ability to initiate and drive tumorigenesis, which contributes to the chance of relapse. So we performed tumorsphere formation assays to explore the effect of JIB04 on tumor initiation (tumorsphere initiating cells viability), growth (primary tumorsphere formation), and relapse (secondary tumorsphere formation) [Supplementary Fig. S5B (representative Lepto1 tumorsphere images), Supplementary Fig. S5C and S5D; Supplementary Fig. S6A–S6C]. To test effects on tumor-initiating capacity, we treated Lepto1–3 cells 48 hours with 0.5 μmol/L JIB04 or DMSO and then cultured the cells to generate tumorspheres 7–10 days. Control cells generated dense and relatively large tumorspheres [Supplementary Fig. S5C and S5D; Supplementary Fig. S6B (representative Lepto1 tumorsphere images; Supplementary Fig. S6C)], but JIB04 pretreated Lepto1 cells were not able to generate many large and dense tumorspheres and most of them remained single. Quantitative analyses based on CCK assays showed that JIB04 treatment significantly decreased the percentage of sphere-initiating cells to 15%–25% of that seen with control cells (Supplementary Fig. S5B and S5C). Next, to determine whether JIB04 inhibits tumorsphere growth, we induced tumorsphere formation for 7 days and then exposed the tumorspheres to 0.5μmol/L JIB04 for 48 hours, followed by FACS sorting and culturing of live (DAPI−) Lepto1 cells derived from variously treated tumorspheres. JIB04 treatment significantly reduced the primary tumorspheres size [Supplementary Fig. S5B and S5D (primary tumorspheres)]. Analyses of these tumorspheres using CCK assays confirmed that JIB04 treatment reduced viability of tumorsphere cells, suggesting that JIB04 inhibits growth of primary tumorspheres [Supplementary Fig. S5B and S5D (secondary tumorspheres)]. To assess effects on relapse, we generated secondary tumorspheres by FACS sorting and culturing surviving (DAPI−) cells from primary tumorspheres cultured in medium without drug for 12 days. Cells derived from DMSO-treated primary tumorspheres formed secondary tumorspheres resembling primary tumorspheres. However, cells derived from JIB04-treated primary tumorspheres did not develop secondary tumorspheres, and the number of viable cells in secondary tumorspheres derived from JIB04-treated primary tumorspheres also decreased (Supplementary Fig. S5B and S5D), implying that tumorsphere regrowth is suppressed by JIB04 pretreatment. Similarly, pretreatment of Lepto2 and 3 cells with 0.5 μmol/L JIB04 for 48 hours not only led to significant reduction in the percentage of sphere-initiating cells to approximately 10% of that seen with control cells but also their ability to develop primary and secondary tumorspheres was also significantly decreased (Supplementary Fig. S6A–S6C).
JIB04 treatment selectively targets the CSC phenotype and functionality of Lepto lines
Next, to determine whether JIB04 antagonizes Lepto lines CSC phenotype and CSC-associated functionality, we performed FACS-based surface expression analysis of CD326+CD49f− and CD44+CD24− on the three Lepto lines pretreated with JIB04. As above, to avoid potential confounding effects of continued JIB04 toxicity, we conducted assays using Lepto1–3 cells pretreated with JIB04 or control DMSO, allowed cells to recover 4 days without drug and then determined the proportions of CD326+CD49f− and CD44+CD24− Lepto1–3 cells (Fig. 3A–C; Supplementary Fig. S7A–S7D). JIB04 pretreatment reduced not only the proportion of living (DAPI−) CD326+CD49f− Lepto1 cells [Fig. 3A (top) and B; Lepto1, Supplementary Fig. S7A and S7B; Lepto2 and 3] but also the proportion of living (DAPI−) CD44+CD24− Lepto1 cells were significantly reduced by approximately 8-fold to 9-fold relative to vehicle [Fig. 3A (bottom) and C; Lepto1, Supplementary Fig. S7C and S7D; Lepto2 and 3]. As a functional measure of CSC frequency, we examined the ability of surviving JIB04-treated CD326+CD49f− and CD44+CD24− Lepto1 cells to form tumorspheres. Specifically, after treating cells with JIB04 (0.25 μmol/L) or DMSO for 48 hours, we FACS-sorted living CD326+CD49f− and CD44+CD24− populations and monitored tumorsphere formation for 10 days without JIB04. JIB04 pretreatment induced an approximately 4-fold to 5-fold decrease in the proportion of tumor-initiating Lepto cells in both populations (Fig. 3D).
JIB04 treatment selectively targets the CSC phenotype and functionality of Lepto lines. A, Representative CD326/CD49f (top) and CD44/CD24 (bottom) FACS profiles of Lepto1 cells pretreated with JIB04 (0.25 μmol/L) and allowed to recover on subsequent days 0 to 3. B, FACS-based quantification of live (DAPI−) CD326+CD49f− Lepto1 cells allowed to recover after 0.25 μmol/L JIB04 pretreatment and then cultured without drug over days 0 to 3, as described in A. Means ± SD of three biological replicates are shown. ***, P < 0.001. C, FACS-based quantification of live CD326+CD49f− Lepto1 cells to quantify the percent of CD44+CD24− Lepto1 cells, which were pretreated with 0.25 μmol/L JIB04 and then cultured in the absence of compounds from day 0 to day 3. Means ± SD of three biological replicates are shown. ***, P < 0.001. D, Quantification of percent of tumorsphere-initiating cells formed from CD326+CD49f− and CD44+CD24− Lepto cells treated as described in B and C. Means ± SD of three technical replicates and three biological replicates are shown. *, P < 0.001. E, Assay for H3K9me3 demethylase activity in lysates of Lepto1 cells treated with 0.2 μmol/L JIB04 demonstrated loss of H3K9me3 demethylase activity in a dose-dependent fashion. Means ± SD of three technical replicates and three biological replicates are shown. ***, P < 0.001. F, Phase-contrast images of live (DAPI−) FACS-sorted CD326+CD49f− and CD44+CD24− Lepto1-GFP cells present in the bottom chamber of Matrigel invasion chambers after treatment with 0.1% DMSO (control) or JIB04 (0.02 or 0.04 μmol/L) for 72 hours. G, Simultaneous differential interference contrast images (at ×10) of Lepto1 cells that had migrated to the bottom chamber of the Matrigel invasion chamber on day 4. Cells had been pretreated with JIB04 (0.04 μmol/L) for 72 hours before being plated in the Matrigel invasion chambers. Scale bar, 50 μm.
JIB04 treatment selectively targets the CSC phenotype and functionality of Lepto lines. A, Representative CD326/CD49f (top) and CD44/CD24 (bottom) FACS profiles of Lepto1 cells pretreated with JIB04 (0.25 μmol/L) and allowed to recover on subsequent days 0 to 3. B, FACS-based quantification of live (DAPI−) CD326+CD49f− Lepto1 cells allowed to recover after 0.25 μmol/L JIB04 pretreatment and then cultured without drug over days 0 to 3, as described in A. Means ± SD of three biological replicates are shown. ***, P < 0.001. C, FACS-based quantification of live CD326+CD49f− Lepto1 cells to quantify the percent of CD44+CD24− Lepto1 cells, which were pretreated with 0.25 μmol/L JIB04 and then cultured in the absence of compounds from day 0 to day 3. Means ± SD of three biological replicates are shown. ***, P < 0.001. D, Quantification of percent of tumorsphere-initiating cells formed from CD326+CD49f− and CD44+CD24− Lepto cells treated as described in B and C. Means ± SD of three technical replicates and three biological replicates are shown. *, P < 0.001. E, Assay for H3K9me3 demethylase activity in lysates of Lepto1 cells treated with 0.2 μmol/L JIB04 demonstrated loss of H3K9me3 demethylase activity in a dose-dependent fashion. Means ± SD of three technical replicates and three biological replicates are shown. ***, P < 0.001. F, Phase-contrast images of live (DAPI−) FACS-sorted CD326+CD49f− and CD44+CD24− Lepto1-GFP cells present in the bottom chamber of Matrigel invasion chambers after treatment with 0.1% DMSO (control) or JIB04 (0.02 or 0.04 μmol/L) for 72 hours. G, Simultaneous differential interference contrast images (at ×10) of Lepto1 cells that had migrated to the bottom chamber of the Matrigel invasion chamber on day 4. Cells had been pretreated with JIB04 (0.04 μmol/L) for 72 hours before being plated in the Matrigel invasion chambers. Scale bar, 50 μm.
Next, to assess effects of JIB04 on histone demethylase activity, we treated Lepto1–3 cells with DMSO or JIB04, made cell lysates, and assayed potential demethylation of an exogenous H3K9me3 peptide substrate to a H3K9me2 product, as described previously (23). We observed a significant (by 30%) reduction in total H3K9me3 demethylase activity in lysates from Lepto cells exposed to 2 μmol/L JIB04 relative to vehicle (Fig. 3E). As a negative control, we observed no inhibition of HDAC activity in lysates, even at high JIB04 doses (Supplementary Fig. S7E and S7F), while HDAC5 activity inhibition was observed in trichostatin A–treated Lepto cells, suggesting that JIB04-mediated targeting of Lepto cells is due in part to its ability to inhibit KDM activity. Interestingly, comparison of JIB04′s effect with that of IOX1, a broad-spectrum inhibitor of 2-oxoglutarate oxygenases, including Jumonji C domain (JmjC) demethylases, suggested that JIB04 is more potent that IOX1 (Supplementary Fig. S7G).
We performed analysis using Matrigel invasion chambers to assess invasiveness of living JIB04-treated, FACS-sorted CD326+CD49f− and CD44+CD24− Lepto1 cells transduced with lentiviruses expressing GFP. We observed a significant decrease in invasiveness potential (based on intensity of GFP present in the bottom chamber) of Lepto1 cells treated with 0.02 or 0.04 μmol/L JIB04 for 48 hours (Fig. 3F and G).
JIB04 reduces HER2+ LC tumor growth in vivo, prolongs overall survival, and decreases histone demethylase activity
We next asked whether JIB04 treatment decreased CSC activity in vivo. To do so, we treated mCherry: LUC-labeled Lepto1, Lepto2, and Lepto3 lines 7 days with JIB04 or DMSO, allowed cells to recover, cultured them for at least 14 days without drug and then injected serial dilutions of cells into NOD/SCID mice via cisternae magna puncture. We then monitored their tumor seeding via BLI-based quantification of LUC over a period of 30 days. JIB04 pretreatment resulted in a >100-fold decrease in tumor-seeding ability relative to DMSO pretreatment for Lepto1, 2, and 3 lines (Fig. 4A).
JIB04 reduces HER2+ LC tumor growth in vivo, prolongs overall survival, and decreases histone demethylase activity. A, Tumor seeding ability of Lepto1, Lepto2, and Lepto3 cells pretreated with DMSO or JIB04. Number of animals with significantly large Lepto-derived tumors were counted per 8 Lepto-injected animals. Comparison between DMSO versus JIB05 pretreated Lepto bearing animal groups is ***, P < 0.001. B, Schematic illustrating implantation and evaluation of mCherry:LUC-expressing Lepto cells (100 K in 20 μL PBS) injected into the cisternae magna of NOD/SCID mice. Mice received intrathecal injections of DMSO or JIB04 (60 or 120 mg/kg, n = 12 per group) on indicated days. Tumor growth was analyzed by BLI and survival by the Kaplan–Meyer method. C, Representative BLI images taken on days 16, 26, 40, and 60 after Lepto1 cell implantation, with or without JIB04 (60 or 120 mg/kg) treatment as per protocol shown in B. D, BLI-based quantification of tumor volume in mouse brain (red) and in the spinal cord (blue) on day 16 post Lepto1 implantation and in the presence or absence of JIB04 treatment, as indicated. Means ± SD; n = 4 (vehicle-treated controls), n = 4 (60 mg/kg JIB04 treated), and n = 4 (120 mg/kg JIB04 treated) mice are shown. ***, P < 0.001. E, BLI-based quantification of tumor volume in mouse brain (red) and in the spinal cord (blue) on day 26 after Lepto1 implantation and in the presence or absence of JIB04 treatment, as indicated. Means ± SD; n = 4 (vehicle-treated controls), n = 4 (60 mg/kg JIB04 treated), and n = 4 (120 mg/kg JIB04 treated) mice are shown. ***, P < 0.001. F, Western blot analysis of histone methylation in FACS-sorted CD326+ Lepto1 cells isolated from control (CTL; day 30), methotrexate-treated (Met; day 30), and JIB04-treated mice on days 30, 40, and 50 post-implantations. Histone3 and H3K4me3 served as loading and internal controls. G, BLI-based quantification of tumor volume from days 10 to 30 after Lepto1 cell implantation. Means of n = 8 (control treated), n = 8 (60 mg/kg JIB04 treated) mice with SDs are shown. ***, P < 0.001. H, Kaplan–Meyer survival curves for each cohort treated as in G. ***, P < 0.01. I, Assay of H3K9me3 demethylase activity in tumor cells isolated from mice treated with JIB04 demonstrated reduction of H3K9me3 demethylase activity. Means ± SD of three technical replicates and three biological replicates are shown. **, P < 0.001.
JIB04 reduces HER2+ LC tumor growth in vivo, prolongs overall survival, and decreases histone demethylase activity. A, Tumor seeding ability of Lepto1, Lepto2, and Lepto3 cells pretreated with DMSO or JIB04. Number of animals with significantly large Lepto-derived tumors were counted per 8 Lepto-injected animals. Comparison between DMSO versus JIB05 pretreated Lepto bearing animal groups is ***, P < 0.001. B, Schematic illustrating implantation and evaluation of mCherry:LUC-expressing Lepto cells (100 K in 20 μL PBS) injected into the cisternae magna of NOD/SCID mice. Mice received intrathecal injections of DMSO or JIB04 (60 or 120 mg/kg, n = 12 per group) on indicated days. Tumor growth was analyzed by BLI and survival by the Kaplan–Meyer method. C, Representative BLI images taken on days 16, 26, 40, and 60 after Lepto1 cell implantation, with or without JIB04 (60 or 120 mg/kg) treatment as per protocol shown in B. D, BLI-based quantification of tumor volume in mouse brain (red) and in the spinal cord (blue) on day 16 post Lepto1 implantation and in the presence or absence of JIB04 treatment, as indicated. Means ± SD; n = 4 (vehicle-treated controls), n = 4 (60 mg/kg JIB04 treated), and n = 4 (120 mg/kg JIB04 treated) mice are shown. ***, P < 0.001. E, BLI-based quantification of tumor volume in mouse brain (red) and in the spinal cord (blue) on day 26 after Lepto1 implantation and in the presence or absence of JIB04 treatment, as indicated. Means ± SD; n = 4 (vehicle-treated controls), n = 4 (60 mg/kg JIB04 treated), and n = 4 (120 mg/kg JIB04 treated) mice are shown. ***, P < 0.001. F, Western blot analysis of histone methylation in FACS-sorted CD326+ Lepto1 cells isolated from control (CTL; day 30), methotrexate-treated (Met; day 30), and JIB04-treated mice on days 30, 40, and 50 post-implantations. Histone3 and H3K4me3 served as loading and internal controls. G, BLI-based quantification of tumor volume from days 10 to 30 after Lepto1 cell implantation. Means of n = 8 (control treated), n = 8 (60 mg/kg JIB04 treated) mice with SDs are shown. ***, P < 0.001. H, Kaplan–Meyer survival curves for each cohort treated as in G. ***, P < 0.01. I, Assay of H3K9me3 demethylase activity in tumor cells isolated from mice treated with JIB04 demonstrated reduction of H3K9me3 demethylase activity. Means ± SD of three technical replicates and three biological replicates are shown. **, P < 0.001.
To determine whether JIB04 alters HER2+ LC growth in vivo, we injected mCherry:LUC-labeled primary Lepto1–3 lines at 100 k density (day 0) into the CSF space of NOD/SCID mice via cisterna magna puncture, treated mice with JIB04 via intraperitoneal injections (60 or 120 mg/kg) or vehicle at days 7, 10, 12, and 14 post-implantation (Scheme; Fig. 4B) and assessed tumor volume based on BLI on days 16 and 26 (Fig. 4C–E and G; Lepto1, Supplementary Fig. S8A and S8B; Lepto2 and 3). Relative to vehicle treatment, JIB04 treatment significantly suppressed growth of tumors derived from Lepto1–3 cells dose dependently [P < 0.001; Fig. 4C–E, and G; Lepto1 and Supplementary Fig. S8A and S8B; Lepto2 and 3). JIB04 treatment resulted in significantly lower tumor growth rates and tumor volume (Fig. 4G; Lepto1 and Supplementary Fig. S8A and S8B; Lepto2 and 3). Moreover, histologic evaluation showed no abnormalities in various organs following treatment (Supplementary Fig. S8E). Kaplan–Meyer analyses demonstrated that JIB04 treatment significantly prolonged survival relative to controls [log-rank (Mantel–Cox test P < 0.01; Fig. 4H; Lepto1 and Supplementary Fig. S8C], with a median survival of 47–51 relative to 18–23 days in untreated controls (Supplementary Fig. S8C). Hematoxylin and eosin analysis showed that the overall number of Lepto1 line–derived lesions in the sagittal brain sections (Supplementary Fig. S8D), coronal brain sections (Supplementary Fig. S9A), horizontal brain sections (Supplementary Fig. S9B), and axial spinal cord sections (Supplementary Fig. S9C) significantly decreased in mice treated with JIB04, relative to vehicle-treated Lepto1 tumor bearing NOD/SCID mice. We also monitored Lepto1-HER2+ LC tumor-bearing NOD/SCID mice (treated with vehicle or JIB04) brain and spinal cord tissue isolates via FACS and documented presence of CD326+mCherry+ Lepto1 in the brain and spinal tissue isolates from vehicle- and JIB04-treated Lepto tumor-bearing mice but the percentage of Lepto1 cells from JIB04-treated mouse brain and spinal cord isolates were significantly lower than the Lepto1 percentage from vehicle-treated Lepto1 tumor-bearing mice (Supplementary Fig. S9D). These results indicate that JIB04 is effective in controlling HER2+ LC tumor growth of Lepto lines in NOD/SCID mouse models.
Given that JIB04 inhibits H3K9me3 demethylase activity in Lepto cells in vitro, we asked whether tumor cells from JIB04-treated mice exhibited decreased histone demethylase activity. To do so, we assayed demethylation of exogenous H3K9me3 substrate in Lepto tumor cell lysates prepared from NOD/SCID mice treated with or without JIB04. JIB04 treatment upregulated trimethylation of both H3K9 and H3K36 relative to that seen in controls, while global H3K4 trimethyl status was not significantly altered, suggesting that JIB04 indeed inhibits KDM histone demethylase family proteins in HER2+ LC tumor cells (Fig. 4F). Moreover, tumor cell lysates from JIB04-treated mice showed reduced total H3K9me3 demethylase activity relative to tumors from vehicle-treated mice (Fig. 4I).
JIB04 inhibits KDM demethylase activity in vitro
JIB04 reportedly inhibits KDM4 and KDM5 enzymatic activities in vitro (23, 32, 33), suggesting that JmjC histone demethylases are JIB04 targets. Thus, we compared potency and specificity of JIB04 inhibition of Jumonji domain–containing histone demethylases by ELISA to quantify demethylated histone substrates. We found that KDM4A, KDM4B, KDM4C, KDM4E, KDM5A, and KDM6B exhibited varying JIB04 sensitivity (Fig. 5A and B): KDM5A was most sensitive (IC50 = ∼300 nmol/L), while KDM6B (IC50 = ∼1 μmol/L) was highly resistant. KDM4 family enzymes had variable IC50 values, ranging between 315 and 345 nmol/L (Fig. 5A and B). TET1, a dioxygenase that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and plays a key role in active DNA demethylation (REF) served as a negative control (Fig. 5C). We also observed strong inhibition of KDM4D activity based on Western blotting of histone substrates (Fig. 5D). Furthermore, cellular thermal shift assays (CETSA) revealed that JIB04 stabilizes KDM4A/4C in Lepto1 cells (Fig. 5E and F) and directly binds to KDM4A and KDM4C in Lepto1 lines. These analyses confirm that JIB04 is a pan-inhibitor of Jumonji domain–containing lysine demethylases.
JIB04 inhibits KDM demethylase activity in vitro. A, JIB04 dose–response curves across Jumonji domain–containing KDM demethylases, as measured by ELISA. Means ± SD of three technical replicates and three biological replicates are shown. IC50 values are given across various experiments; n = 3 for KDM4A, KDM4B, KDM4C. B, JIB04 dose–response curves across Jumonji domain–containing KDM demethylases as measured by ELISA. Means ± SD of three technical replicates and three biological replicates are shown. IC50 values are given across various experiments; n = 3 for KDM4E, KDM5A, and KDM6B. C, JIB04 selectively inhibits Jumonji enzymes over other cellular hydroxylases in vitro. Mean of three for TET1 are shown, with SDs. D, Direct measure of histone demethylation by Western blot analysis (left, KDM4D) showed inhibition of Jumonji enzyme activity by JIB04. E, Cellular thermal shift assay demonstrated that KDM4A in Lepto1 cells are stabilized post JIB04 treatment. Representative immunoblots from CETSA experiments. F, Cellular thermal shift assay demonstrated that KDM4C in Lepto1 cells are stabilized after JIB04 treatment. Representative immunoblots from CETSA experiments.
JIB04 inhibits KDM demethylase activity in vitro. A, JIB04 dose–response curves across Jumonji domain–containing KDM demethylases, as measured by ELISA. Means ± SD of three technical replicates and three biological replicates are shown. IC50 values are given across various experiments; n = 3 for KDM4A, KDM4B, KDM4C. B, JIB04 dose–response curves across Jumonji domain–containing KDM demethylases as measured by ELISA. Means ± SD of three technical replicates and three biological replicates are shown. IC50 values are given across various experiments; n = 3 for KDM4E, KDM5A, and KDM6B. C, JIB04 selectively inhibits Jumonji enzymes over other cellular hydroxylases in vitro. Mean of three for TET1 are shown, with SDs. D, Direct measure of histone demethylation by Western blot analysis (left, KDM4D) showed inhibition of Jumonji enzyme activity by JIB04. E, Cellular thermal shift assay demonstrated that KDM4A in Lepto1 cells are stabilized post JIB04 treatment. Representative immunoblots from CETSA experiments. F, Cellular thermal shift assay demonstrated that KDM4C in Lepto1 cells are stabilized after JIB04 treatment. Representative immunoblots from CETSA experiments.
Modulation of KDM demethylase family enzyme levels alters JIB04 effects in Lepto cells
Given that JIB04 inhibits KDM4A and KDM4C demethylase activity in Lepto1 cells and lysates (Fig. 3E, 4F, and 4I), we asked whether KDM4A and KDM4C play a role in development of HER2+ LC tumors. To do so, we analyzed KDM4A/4C expression in Lepto1-derived HER2+ LC tumors from the axial spinal cord sections of Lepto1 tumor-bearing NOD/SCID mice. Both KDM4A and KDM4C were significantly overexpressed in Lepto1-derived HER2+ LC tumors compared with adjacent nontumor neural tissues (Fig. 6A). We also observed significant overexpression of KDM4A/4C in Lepto1, 2, and 3 lines and other HER2+ lines (BT-474 and SK-BR3) relative to HER2− lines (MDA-MB-231, T47D, and MCF7) or to the noncancerous MCF10A breast epithelial cell line (Fig. 6B). Evaluation of genome-wide CRISPR and short hairpin RNA (shRNA) screens revealed KDM2A, KDM8, and KDM1A to be essential in all breast cancer lines analyzed, suggesting they are not targets for HER2+ LC and would not make good therapeutic targets for HER2+ LC (Supplementary Fig. S10A and S10B). These findings, along with analyses of KDM4A and KDM4C coexpression in various metastatic breast cancer lines using the Cancer Dependency Map (DepMap) portal (Fig. 6C; Supplementary Table S5) and dependency data from the CRISPR and shRNA screens, prompted us focus on KDM4A/4C function in HER2+ LC.
Modulation of KDM demethylase family enzyme levels alters JIB04 effects in Lepto cells. A, IF analysis of serial spinal cord sections taken from mice 10 days after Lepto1 cell implantation. Imaging of OPCs (Olig2, purple), KDM4A or KDM4C (green), and mCherry (Lepto1 cells, red) indicates KDM4A (top) and KDM4C (bottom) overexpression in the Lepto1 cell layer relative to surrounding neuronal tissues. B, Quantification of KDM4A (left) and KDM4C (right) transcript levels as indicated by qRT-PCR. Color coding indicates relative levels of HER2. Means ± SD of three technical replicates and three biological replicates are shown. C, DepMap-based analysis of KDM4A/4C coexpression in metastatic breast cancer cell lines; listed in Supplementary Table S2. D, Heatmap demonstrating fold change in number of viable Lepto1 cells following doxycycline (DOX)-induced shKDM4A, shKDM4C, or shGFP expression. Means ± SD of three technical replicates and three biological replicates are shown. KDM4A or KDM4C knockdown in Lepto1 cells significantly decreased the doubling time, relative to uninduced and shGFP control Lepto1 cells. E, FACS-based analyses of Annexin-V positivity in Lepto1 cells from days 0 to 6 after doxycycline treatment to induce shKDM4A (left) or shKDM4C (right) expression. Control cells were uninduced. Means ± SD of three biological replicates are shown. *, P < 0.001. F, Western blot analysis of KDM4A/4C protein levels 48 hours after doxycycline (0 to 5 μg/mL) treatment to induce shKDM4A and shKDM4C in Lepto1 cells, as a means to confirm knockdown. G, Representative FACS plots of Annexin-V positivity in CD326+ Lepto1 cells with or without doxycycline-induced expression of shKDM4A (left) or shKDM4C (right).
Modulation of KDM demethylase family enzyme levels alters JIB04 effects in Lepto cells. A, IF analysis of serial spinal cord sections taken from mice 10 days after Lepto1 cell implantation. Imaging of OPCs (Olig2, purple), KDM4A or KDM4C (green), and mCherry (Lepto1 cells, red) indicates KDM4A (top) and KDM4C (bottom) overexpression in the Lepto1 cell layer relative to surrounding neuronal tissues. B, Quantification of KDM4A (left) and KDM4C (right) transcript levels as indicated by qRT-PCR. Color coding indicates relative levels of HER2. Means ± SD of three technical replicates and three biological replicates are shown. C, DepMap-based analysis of KDM4A/4C coexpression in metastatic breast cancer cell lines; listed in Supplementary Table S2. D, Heatmap demonstrating fold change in number of viable Lepto1 cells following doxycycline (DOX)-induced shKDM4A, shKDM4C, or shGFP expression. Means ± SD of three technical replicates and three biological replicates are shown. KDM4A or KDM4C knockdown in Lepto1 cells significantly decreased the doubling time, relative to uninduced and shGFP control Lepto1 cells. E, FACS-based analyses of Annexin-V positivity in Lepto1 cells from days 0 to 6 after doxycycline treatment to induce shKDM4A (left) or shKDM4C (right) expression. Control cells were uninduced. Means ± SD of three biological replicates are shown. *, P < 0.001. F, Western blot analysis of KDM4A/4C protein levels 48 hours after doxycycline (0 to 5 μg/mL) treatment to induce shKDM4A and shKDM4C in Lepto1 cells, as a means to confirm knockdown. G, Representative FACS plots of Annexin-V positivity in CD326+ Lepto1 cells with or without doxycycline-induced expression of shKDM4A (left) or shKDM4C (right).
To assess whether antiproliferation effects of JIB04 in Lepto lines were due to KDM4A/4C inhibition, we cloned shRNAs targeting KDM4A or KDM4C into pTRE3G-ZsGreen1, a doxycycline-inducible lentiviral vector. Doxycycline treatment of Lepto1 cells transduced with either shKDM4A-GFP or shKDM4C-GFP lentiviruses resulted in highly specific knockdown of respective targets and significantly decreased Lepto1 cell proliferation (Fig. 6D and F). KDM4A or KDM4C knockdown also increased Annexin-V positivity in Lepto 1 cells by day 6 after doxycycline administration (Fig. 6E and G), phenocopying JIB04 treatment. Concurrently, we also achieved highly specific esiRNA-mediated knockdown of KDM4A–E, KDM6B, and KDM5A in Lepto1 cells, as confirmed by qRT-PCR and Western blot analysis (Supplementary Fig. S10C and S10D). Interestingly, knockdown of KDM4A or KDM4C, but not of other KDM4 or KDM5 family members tested, significantly and increased apoptosis, based on FACS-based quantification of Annexin-V positivity and reduced Lepto1 cell viability (Supplementary Fig. S10E and S10F), again phenocopying JIB04 treatment. KDM5A depletion also reduced cell viability and augmented cell surface Annexin-V expression (Supplementary Fig. S10E and S10F). However, relative to effects of KDM4A or KDM4C knockdown, those phenotypes were weaker. In addition, KDM4A and KDM4C protein levels were also higher in Lepto 2 and Lepto3 cells relative to other breast cancer lines (Supplementary Fig. S11A). Interestingly, Kaplan–Meier survival plots based on data from the cBioPortal showed that patients with breast cancer with higher KDM4A/4C expression had shorter overall survival (124.13 months) than patients with lower levels (161.93 months; log-rank test P < 0.001; Supplementary Fig. S11B). shRNA-mediated conditional knockdown of KDM4A and/or KDM4C also significantly affected the CD44+CD24− CSC phenotype of Lepto1 cells (Supplementary Fig. S11C). Knockdown of KDM4A and/or KDM4C led to approximately 2- to 3-fold reduction in the CSC phenotype of Lepto1 cells, suggesting that KDM4A and KDM4C maintain CSC phenotypes in the Lepto cells. Indeed, horizontal brain sections of uninduced and doxycycline-induced (shKDM4A and shKDM4C overexpression) Lepto1 tumor-bearing NOD/SCID mice demonstrate that depletion of KDM4A and KDM4C leads to significant reduction in Lepto1-induced tumor volume (Supplementary Fig. S11D). Furthermore, Lepto cells conditionally overexpressing shKDM4A and/or KDM4C required significantly higher number of cells for tumor seeding in NOD/SCID mice models (Supplementary Fig. S11E).
Next, we cloned KDM4A and KDM4C open reading frames into pTRE3G-ZsGreen1 to establish Tet-On 3G tetracycline-inducible expression constructs in Lepto1–3 lines. Infection efficiency and thus overexpression following doxycycline treatment was quantified via the ZsGreen signal in flow cytometry analysis. Doxycycline-mediated KDM4A or KDM4C overexpression was confirmed by Western blot analysis (Supplementary Fig. S12A and S12C). Cell viability was comparable in both uninduced (−doxycycline) and induced (+doxycycline) KDM4A or KDM4C-overexpressing Lepto lines, and all showed normal cell growth kinetics (Supplementary Fig. S12B and S12D; red trendline). However, following exposure of Lepto1 lines to 0.5 μmol/L JIB04, induced KDM4A- or KDM4C-overexpressing lines showed significant rescue of viability relative to uninduced lines (Supplementary Fig. S12B and S12D; blue trendline). Comparable phenotypes were also seen in Lepto2 and 3 lines (Supplementary Fig. S13A–S13D).
Next, we evaluated effects of conditional KDM4A and/or KDM4C depletion on Lepto1 cell self-renewal capacity (namely, the ability to survive and form colonies from single cells). We found that either KDM4A and/or KDM4C depletion or JIB04 (0.01–0.1 μmol/L) pretreatment dramatically reduced clonogenic proliferation of Lepto1, 2, and 3 lines over a period of 10 days (Fig. 7B, D, and E; Lepto1 and Supplementary Fig. S14A–S14D; Lepto2 and 3), strongly suggesting that JIB04′s antiproliferative effect is in part a direct result of Jumonji activity inhibition.
KDM4A/4C regulates GMCSF expression in HER2+ LC cells. A, Quantification of GMCSF levels in conditioned media from Lepto1 cells cultured in media supplemented with hCSF, with or without JIB04. Data are based on Cytokine XL array blots shown in Supplementary Fig. S12E. Additional secreted factors are listed in Supplementary Table S3. B, Top, scheme showing lentiviral Tet-On 3G inducible GMCSF expression cassette used in this study for conditional depletion of KDM4A/4C. Bottom, Western blot analyses of KDM4A and KDM4C protein levels in Lepto1 cells expressing shKDM4A and/or shKDM4C following 2 and 5 μg/mL doxycycline (DOX) induction. C, ELISA-based quantification of GMCSF in culture media of Lepto1 cells with or without shRNA-mediated depletion of KDM4A and/or KDM4C (n = 3). Control cells were not treated with doxycycline. ***, P < 0.001. D, Top row, clonogenic proliferation of Lepto1 cells with or without doxycycline-mediated depletion of KDM4A and/or KDM4C. Bottom row, similar analyses of untransduced Lepto1 cells treated with DMSO (control) or 0.01 to 0.1 μmol/L JIB04. In both analyses, cells were stained with crystal violet. Means ± SD of three technical replicates and three biological replicates are shown. Image of whole well from a 6-well plate is shown. E, Quantification of colonies depicted in D. Means ± SD of three technical replicates and three biological replicates are shown. ***, P < 0.001. F, Quantification of activity of a GMCSF luciferase reporter in primary Lepto1 cells with or without doxycycline-mediated depletion of KDM4A/4C or treated with DMSO or JIB04. Means ± SD of three technical replicates and three biological replicates are shown. ***, P < 0.001.
KDM4A/4C regulates GMCSF expression in HER2+ LC cells. A, Quantification of GMCSF levels in conditioned media from Lepto1 cells cultured in media supplemented with hCSF, with or without JIB04. Data are based on Cytokine XL array blots shown in Supplementary Fig. S12E. Additional secreted factors are listed in Supplementary Table S3. B, Top, scheme showing lentiviral Tet-On 3G inducible GMCSF expression cassette used in this study for conditional depletion of KDM4A/4C. Bottom, Western blot analyses of KDM4A and KDM4C protein levels in Lepto1 cells expressing shKDM4A and/or shKDM4C following 2 and 5 μg/mL doxycycline (DOX) induction. C, ELISA-based quantification of GMCSF in culture media of Lepto1 cells with or without shRNA-mediated depletion of KDM4A and/or KDM4C (n = 3). Control cells were not treated with doxycycline. ***, P < 0.001. D, Top row, clonogenic proliferation of Lepto1 cells with or without doxycycline-mediated depletion of KDM4A and/or KDM4C. Bottom row, similar analyses of untransduced Lepto1 cells treated with DMSO (control) or 0.01 to 0.1 μmol/L JIB04. In both analyses, cells were stained with crystal violet. Means ± SD of three technical replicates and three biological replicates are shown. Image of whole well from a 6-well plate is shown. E, Quantification of colonies depicted in D. Means ± SD of three technical replicates and three biological replicates are shown. ***, P < 0.001. F, Quantification of activity of a GMCSF luciferase reporter in primary Lepto1 cells with or without doxycycline-mediated depletion of KDM4A/4C or treated with DMSO or JIB04. Means ± SD of three technical replicates and three biological replicates are shown. ***, P < 0.001.
KDM4A/4C regulates GMCSF expression in HER2+ LC cells
To determine how KDM4A or KDM4C activity enables HER2+ LC tumor growth, we cultured Lepto1 cells with or without JIB04 and analyzed cell-free growth media from each sample using a Cytokine XL array to decipher the alterations in the secretome of the Lepto1 culture upon JIB04 exposure. We observed significantly higher GMCSF levels in growth media from Lepto1 cells cultured without JIB04 relative to those grown in the presence of JIB04 (Fig. 7A; Supplementary Fig. S12E; Supplementary Table S6). To assess potential effects of KDM4A/4C expression on GMCSF expression, we conditionally knocked down either KDM4A or KDM4C alone or together in all three Lepto lines cells using doxycycline-inducible shKDM4A and/or shKDM4C (Fig. 7B; Supplementary Fig. S14A). ELISA-based analysis of cell culture media demonstrated that KDM4A and/or KDM4C depletion significantly reduced GMCSF protein levels in all three lines, relative to uninduced controls, phenocopying JIB04 treatment (Fig. 7C; Lepto1 and Supplementary Fig. S14A and S14E; Lepto2 and 3). Next, to determine whether KDM4A and/or KDM4C deficiency altered GMCSF transcription, we transfected Lepto1, 2, or 3 cells with a luciferase reporter construct driven by the GMCSF proximal promoter (+1 to −500 bp) and treated cells with either with JIB04 or doxycycline, the latter to induce shKDM4A-GFP or shKDM4C-GFP expression. Luciferase activity was significantly reduced relative to control groups in all treatment groups [Fig. 7F; Lepto1 and Supplementary Fig. S14F (Lepto2 and 3)], suggesting that GMCSF transcription may be regulated by both KDM4A/4C.
Discussion
Management of HER2+ LC tumors in the clinic requires a multidisciplinary combinatorial approach including radiation and intrathecal therapy, but response rates to such treatments remain <20% (34–37). Thus, there is an urgent need to develop effective therapies that specifically target HER2+ LC. Recent studies have demonstrated improved efficacy of trastuzumab treatment in all stages of breast cancer relative to other drugs tested so far, and there has been interest in administering trastuzumab intrathecally for management of HER2+ LC (24, 38–41) Other than trastuzumab, no other drug has been shown capable of improving outcomes, and identifying candidate therapeutics for HER2+ LC is hampered by the absence of in vitro and in vivo models that faithfully recapitulate the human disease (28). As a result, efforts to develop relevant pharmacologic targeting of epigenetic factors have had limited success. To overcome this barrier, we derived three novel primary HER2+ LC lines, Lepto1, Lepto2, and Lepto3, from human HER2+ LC tumor nodules. Our in vivo analyses of all three lines demonstrated that intrinsically they maintain a spatial preference for the leptomeningeal surface, as observed in human patients (Fig. 1C–E; Lepto1 and Supplementary Fig. S1A–S1C; Lepto2 and Lepto3). In addition, intracardiac injection of the Lepto lines into NOD/SCID mice led to systemic metastasis, including within the brain and spinal cord (Supplementary Fig. S2A–S2C). Lepto cells also formed metastases in various organs and produced significant brain (Supplementary Fig. S2C) and spinal cord metastases in NOD-SCID mice. These findings suggest that Lepto lines successfully form metastatic colonies in a brain and leptomeningeal microenvironment.
Furthermore, our detailed characterization of the Lepto1 cell surface revealed homogeneous expression of CD326+, which is reportedly highly expressed in HER2+ LC tumors and has been used as a marker to detect tumor cells in CSF of patients with HER2+ LC (42, 43). Interestingly, Lepto1 cells developed here exhibit a CD44+CD24− phenotype, which has been previously been ascribed to CSC populations (44). As a functional measure of CSC frequency, we measured resistance of Lepto1 cells, relative to other metastatic and/or HER2+ breast cancer lines, to various drugs. Consistent with their CSC phenotype, Lepto1 cells were more resistant than the other lines to several drugs used to treat HER2+ LC. We also observed that, compared with other breast cancer lines, Lepto1 cells were able to form larger tumorspheres in vitro more efficiently and demonstrated greater tumor-seeding efficiency in vivo.
Using our bona fide HER2+ LC primary Lepto cell lines, we then conducted an unbiased epigenetic drug screen to systematically identify drugs that specifically target HER2+ LC tumors. That screen identified JIB04 as a specific inhibitor of HER2+ LC growth in vitro and in vivo. JIB04 is a pan-selective inhibitor of Jumonji demethylases previously reported to possess anticancer functionality (45, 46). For example, several groups have demonstrated that JIB04 inhibits growth of drug-resistant lung cancer and glioblastoma cells and induces their apoptosis (24, 45–48). Another recent study reports that JIB04 antagonizes survival and maintenance of CSCs in colorectal carcinoma cell lines (33). However, there are no reports of JIB04 effects on HER2+ LC. We found that JIB04 markedly and preferentially reduced Lepto1 and Lepto2 cell viability and significantly decreased the proportion of cells exhibiting the representative CD44+CD24− CSC phenotype time dependently. Consistently, JIB04 treatment reduced Lepto1 cell self-renewal and tumor-initiating capacity. Furthermore, JIB04 treatment reduced formation of both primary and secondary tumorspheres, supporting the idea that it markedly antagonizes CSC activity and alters Lepto1 cell phenotypes. In vivo, JIB04 treatment effectively reduced the size of Lepto-derived xenograft tumors in NSG mice. Collectively, our results demonstrate that JIB04 has potent and selective antiproliferative effects on primary Lepto cells and can effectively inhibit their stemness and invasive properties, suggesting that it could be used to specifically target HER2+ LC tumors, which are enriched with CSCs. Several reports have demonstrated that JIB04 treatment modulates many molecular functions and pathways, including those underlying several cancers (24, 32, 33, 47, 49). In particular, these findings suggest that JIB04 has anti-CSC activity in solid tumors and in hematologic malignancies. Importantly, JIB04 reportedly alters Wnt/β-catenin signaling, which has recently been shown to regulate expression of genes associated with CSCs as well as with the epithelial-to-mesenchymal transition (23, 33).
Our study establishes an additional mechanism through which JIB04 specifically targets HER2+ LC. We observed that JIB04 treatment increased two different histone modifications, H3K9me3 and H3K36me3, which are the main targets of the KDM family of histone demethylases. Indeed, others have demonstrated that JIB04 inhibits the KDM4 family of proteins, and we confirmed that JIB04 antagonizes their activity. Furthermore, cellular thermal shift assays demonstrated direct engagement of JIB04 with KDM4A and KDM4C in Lepto1 lines. To determine whether JIB04-mediated decreases in cell viability, cell-cycle arrest, and induction of apoptosis are a direct consequence of Jumonji enzyme inhibition, we knocked down various Jumonji domain–containing lysine demethylases and found that depletion of only KDM4A and KDM4C significantly decreased Lepto cell viability. Moreover, as expected, doxycycline-dependent conditional overexpression of KDM4A/4C resulted in partial rescue of JIB04-induced loss of cell viability. This outcome could be due to upregulation of C-MYC and its target genes in Lepto1 cells, which would likely promote proliferative signaling. These findings establish that JIB04′s transcriptional and anti-proliferative activity is at least in part the result of Jumonji inhibition. To further assess KDM4A/4C function in HER2+ LC, we calculated essentiality scores of various Jumonji family lysine demethylases (Supplementary Fig. S10A and S10B) and found that, unlike KDM2A and KDM8 (which are pan-essential), KDM4A and KDM4C are “target-essential,” meaning that their overexpression represents a particular HER2+ LC vulnerability. Interestingly, KDM4A and KDM4C are highly expressed in other HER2+ breast cancer lines; thus, treatment with JIB04 or other KDM4A/4C inhibitors may be effective against those cancers as well.
We also demonstrated that JIB04 treatment, as well as KDM4A and/or KDM4C depletion, impaired clonogenic proliferation of the three Lepto lines, most likely by reducing their expression and secretion of GMCSF, which we previously identified as an autocrine signaling driver of HER2+ LC growth (50). Furthermore, activity of a luciferase reporter driven by the GMCSF promoter decreased in KDM4A/4C-depleted relative to control in all the three Lepto cell lines tested. One model that may account for these outcomes is suggested previously (51, 52): these reports demonstrate that NFκB not only binds to KDM4A and KDM4C but serves as an inducible transcriptional activator of GMCSF via sequence-specific interaction with the GMCSF promoter (52). Thus, KDM4A/4C may modulate GMCSF promoter activity in the context of Lepto1 cells through interaction with NFκB. However, further investigation is warranted. Interestingly, shRNA-mediated KDM4A/KDM4C depletion did not prevent growth of Lepto cells in spinal cord. However, JIB04-treated Lepto tumor-bearing NOD/SCID mice had less growth in the spinal cord. These suggest that JIB04 may also target other signaling pathways necessary for the regulation of Lepto cell migration to the spinal cord.
In summary, we have established much-needed Lepto cell lines that model HER2+ LC and used them to show that these cells require KDM4A/4C most likely to maintain GMCSF expression that drives tumor growth in vivo and in vitro. Thus, our work provides the necessary impetus to develop targeted inhibitors of KDM4A/KDM4C for clinical treatment of HER2+ LC and potentially other HER2+ breast cancers in which KDM4A/KDM4C are highly expressed.
Authors' Disclosures
No disclosures were reported by the authors.
Authors' Contributions
A. Bhan: Conceptualization, resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. K.I. Ansari: Resources, data curation, formal analysis. M.Y. Chen: Conceptualization, resources, data curation, software, formal analysis, supervision, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. R. Jandial: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, writing–original draft, project administration, writing–review and editing.
Acknowledgments
The authors express their gratitude to the City of Hope Analytical Cytometry Core Facility. This work was made possible by the generous support of the City of Hope Department of Surgery and a grant from the U.S. Department of Defense Breast Cancer Research Program (W81XWH-19-1-0310).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.