Inhibition of NF-κB DNA Binding Suppresses Myeloma Growth via Intracellular Redox and Tumor Microenvironment Modulation

Multiple myeloma is the second most common hematologic malignancy, with an incidence of about 32,000 new cases and almost 13,000 deaths per year in the United States. Current first-line therapies include proteasome inhibitors, immunomodulators such as lenalidomide, alkylating agents, monoclonal antibodies such as daratumumab, and steroids. Several phase III trials have established the use of high-dose chemotherapy followed by autologous stem cell transplantation as standard of care in eligible patients (1). Second-generation proteasome inhibitors as well as third-generation immunomodulators have contributed to improving outcomes in the previous decade (2), and B-cell maturation antigen (BCMA)-targeted agents have shown promise in relapsed or refractory multiple myeloma (3). However, almost all patients eventually become refractory to currently available therapies. Therefore, there is an urgent need for novel agents with enhanced efficacy in this setting.

The NF-κB family of transcription factors is found in all nucleated cells. This pathway plays an important role in B-cell proliferation, T-cell activation, regulation of inflammatory responses, as well as the pathogenesis of cancer (4). The NF-κB family comprises of 5 structurally related monomeric subunits: Rel A, Rel B, c-Rel, p50, and p52. The combinatorial association of these subunits produces transcription factors with dimerization and DNA binding domains. In latent form, NF-κB is bound to an inhibitory protein (IκB). When stimulated extracellularly (via ligands such as TNFα, IL6, RANKL, etc.), NF-κB dimers translocate into the nuclei and mediate the expression of a plethora of immune/ stress response genes and pro-survival factors (4). NF-κB is constitutively active in multiple myeloma cells (5, 6) promoting cell proliferation, survival and resistance to chemotherapy (7). Consequently, NF-κB is an attractive therapeutic target in multiple myeloma, with both canonical and noncanonical NF-κB pathways contributing to the total NF-κB activity. NF-κB also regulates transcriptional signatures involved in inflammation and immunity, including the programmed death-ligand 1 (PD-L1)/programmed death protein 1 (PD-1) axis (8). Inhibition of NF-κB DNA binding in multiple myeloma might therefore not only counteract upstream activating mutations but also modulate the immunosuppressive tumor microenvironment (TME). PD-L1/PD-1 interaction results in suppression of T cell–mediated cytotoxicity (9), promoting an immunosuppressive milieu in TME (10). Disruption of the PD-L1/PD-1 axis by monoclonal antibody therapy has been highly effective in several solid tumors but has shown only limited activity against multiple myeloma in initial trials (11). This has led to the additional investigation of protocols combining immune checkpoint blockade with lenalidomide or dexamethasone (12, 13). After initial setbacks, immunotherapeutic agents have now emerged as an attractive option for the treatment of multiple myeloma, but additional preclinical and clinical investigations are warranted to establish safe and effective treatment regimens (14) such as the combination of NF-κB inhibition with immune checkpoint blockade. Here, we introduce IT848, a small molecule inhibitor of NF-κB DNA binding with drug-like physicochemical properties, as a candidate for future clinical development of an NF-κB inhibitor drug. We show that IT848 combines NF-κB inhibition with dysregulation of the redox homeostasis in multiple myeloma cells as well as favorable immunomodulatory properties, translating into significant in vitro and in vivo efficacy against multiple myeloma.

We previously identified thiohydantoin (IT603) and naphthalenethiobarbiturate (IT901) derivatives as conformation-disrupting direct NF-κB inhibitors. After additional structure activity relationship analysis and pharmacokinetics (PK) studies, the novel small molecule IT848 [9-chloro-8-(hexyloxy)-2H-chromeno(2,3-d)pyrimidine-2,4(3H)-dione], molecular weight = 348.78 g/mol was selected as our drug candidate. The IT848 synthesis method is described in detail in our patent (see example # 21, Table 1 of ref. 15).

NOD/SCID/IL2Rγ(null) (NSG) mice were obtained from the Jackson Laboratory for xenograft experiments of multiple myeloma. MM.1S human multiple myeloma cells were transduced with dtomato-firefly luciferase and 5×10⁶ cells were administered to NSG mice via tail vein injection. C57Bl/KaLwRij mice were originally obtained from Envigo (Horst, the Netherlands) and maintained at the Center for Discovery and Innovation. All animal studies were conducted in accordance with Institutional Animal Care and Use Committee (IACUC) standards and with approval of the IACUC of the CDI (protocol#291).

Mice used for these experiments were 6 to 9 weeks old. For the study in immunocompetent mice, 5TGM1 mouse multiple myeloma cells (16) were transduced with dtomato-firefly luciferase and 1.5×10⁶ cells were administered to C57Bl/KaLwRij mice via tail vein injection. Bone marrow engraftment of multiple myeloma cells was confirmed by in vivo bioluminescence imaging (BLI) prior to initiation of treatment, and multiple myeloma progression was monitored at the indicated time points by in vivo BLI. Briefly, mice were anesthetized using isoflurane inhalant anesthetic and administered 3-mg/kg firefly luciferin intraperitoneally and imaged using the IVIS Lumina X5 optical imaging platform. We superimposed pseudocolor images showing the whole-body distribution of bioluminescent signal intensity on grayscale photographs and quantified total flux (photons s⁻¹) for individual mice using Living Image 4.7.3 software (Perkin Elmer, Waltham, MA). In addition, coelenterazine was used to detect changes in regional reactive oxygen species production in vivo in response to IT848 administration (17). For in vivo efficacy studies, IT848 was dissolved in PBS with 30% (w/w) cyclodextrin and 5% PEG 1000 to prepare a 1-mg/mL solution. Mice received either empty vehicle solution or 10-mg/kg body weight of IT848 solution via intraperitoneal injection on either 3 days/week for 4 weeks or on 5 days/week for 2 weeks.

Vendor-authenticated human multiple myeloma cell lines U266, MM.1S, and NCI-H929 were originally obtained from the ATCC (Manassas, VA) in 2018. The human diffuse large B-cell lymphoma (DLBCL)-derived cell lines U2932, HBL1, and TMD8 were kindly provided by Dr. Anas Younes (Memorial Sloan Kettering Cancer Center) in 2013 and authenticated before using them by the MD Anderson Characterized Cell Lines Core Facility. These cells were cultured in RPMI1640 medium supplemented with 10% to 20% heat-inactivated FBS (GIBCO BRL, Gaithersburg, MD), 1% l-glutamine, and penicillin–streptomycin in a humid environment of 5% CO₂ at 37°C. The 5TGM1 cell line was kindly provided by Dr. Rena Feinman (Center for Discovery and Innovation, Nutley, NJ). Vendor-authenticated NIH 3T3 cells were purchased from ATCC in 2019.

A vendor-authenticated NF-κB/Jurkat/GFP transcriptional reporter cell line (18) was purchased from System Biosciences (Mountain View, CA) in 2015. Cells were stimulated with human TNFα (10 ng/mL; Gemini Bio Products, West Sacramento, CA) and incubated in the presence of IT848 or empty vehicle. NF-κB activity (GFP fluorescence intensity) was analyzed by flow cytometry. Vendor-authenticated ARE/HepG2/Luciferase reporter cells and Nuclear factor of activated T-cells (NFAT)/Jurkat/luciferase transcriptional reporter cells were purchased from BPS Bioscience (San Diego, CA) in 2017. ARE/Nrf2 and NFAT transcriptional activity was determined using the One-Step Luciferase Assay System (BPS Biosciences, San Diego, CA). Luminescence was measured with a multimodal plate reader (Infinite 200 pro from Tecan, Switzerland). Human Epstein-Barr Virus (EBV)-transformed B lymphoblastoid cells were generated from peripheral blood mononuclear cell (PBMC) of a healthy donor and kindly provided by Richard O'Reilly (Memorial Sloan Kettering Cancer Center, NY) in 2018. All cell lines were used for experiments within 3 weeks of thawing (up to 4 passages) and were annually tested for Mycoplasma negativity using a Mycoplasma PCR Kit (MycoAlert Kit, Lonza).

Human PBMCs were purchased from United States Biological (Salem, MA). Human multiple myeloma patient cells were collected after informed written consent was obtained, using Institutional Review Board protocol Pro00004259 at Hackensack Meridian Health–John Theurer Cancer Center. All studies of patient materials were conducted in accordance with the Declaration of Helsinki.

MM.1S and 5TGM1 cells were transduced using 3rd generation lentiviral vector with bi-cistronic expression of dtomato and luciferase (pUltra-Chili-Luc; Addgene #48688). Transduced cells were expanded and individual clones/population with high dtomato expression were sorted using a BD FACSMelody. Bioluminescence signal intensity of single clones was determined, and the brightest clones were used for subsequent experiments.

RNA was purified using an AllPrep DNA/RNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA quantity was measured with a Nanodrop spectrophotometer (DeNovix, Wilmington, DE). RNA was submitted to Genewiz (South Plainfield, NJ) for cDNA library creation and sequencing. RNA sequencing (RNA-seq) was performed in triplicate for each treatment condition on an Illumina Hi-Seq 2×150 bp configuration, single index, per lane with estimated data output as ∼350 M raw paired-end reads per lane (19). Initial sample quality control involved assessing RNA integrity and library size by Agilent Tapestation and concentration by Qubit assay, and final quantitation by qPCR. Differential expression analysis between sets of conditions was performed using the DESeq2 pipeline of Basepair analysis platform (Basepair, New York, NY). Gene set enrichment analysis was performed as previously described (20) and a visualization of the canonical NF-κB pathway from the KEGG database is presented.

The data generated in this study are available within the article and its Supplementary Data files. The RNA-seq data generated in this study are publicly available in Gene Expression Omnibus at GSE200385.

We previously described a novel small molecule compound mediating anticancer properties by combining inhibition of NF-κB transcriptional activity with the capacity to modulate the redox balance of B-cell lymphoma cells (21). After additional structure activity relationship analysis and in vivo drug metabolism and PK testing of our lead molecules, IT848 emerged as the most suitable candidate for drug development (Fig. 1A). In silico molecular docking analysis (22) identified IT848 binding sites on all NF-κB subunits (Fig. 1B), and homology modeling indicated that IT848 interacts with thiol groups of cysteines of the Rel/NF-κB subunits. IT848 elicited strong and dose-dependent inhibition of global NF-κB transcriptional activity both in lymphoma (Jurkat, TMD8) and myeloma (U266) cell lines or primary cells (Epstein–Barr virus—transformed B lymphoblastoid cells; Fig. 1C and D). Moreover, we confirmed dose-dependent target-specific in vivo activity of IT848 (Supplementary Fig. S1). Western blot analysis of NF-κB complex proteins [IKΚBeta, IκBalpha, interferon regulatory factor 4 (IRF4)] further validated IT848 as a potent NF-κB inhibitor with consistent, inhibitory effects across several DLBCL and multiple myeloma cell lines (Fig. 1E). Analysis of the NF-κB target gene and pro-tumorigenic cytokine IL6 (23, 24) in representative DLBCL and multiple myeloma cell lines by RT-qPCR (Fig. 1F) and enzyme-linked immunosorbent assay (ELISA; Fig. 1G) demonstrated IT848 mediated supression of IL6 expression, an effect that might not only be useful therapeutically, but also as a biomarker for IT848 activity in multiple myeloma cells. To further elucidate the mechanism of action of IT848 and confirm its capacity to act as intended as a direct NF-κB inhibitor, we assessed DNA binding of individual NF-κB subunits in response to IT848 treatment. We used MicroScale Thermophoresis to characterize binding affinities of IT848 to c-Rel and p50, and to analyze DNA binding of c-Rel and p50 following IT848 treatment. We found that IT848 did not bind to DNA, but it showed binding affinity to both c-Rel and p50 in the low μmo/L range (K_d of 3.9 and 7.0, respectively). In addition, IT848 treatment disrupted DNA binding of c-Rel and decreased p50 DNA binding 30 to 100-fold (Supplementary Fig. S2). In line with this, NF-κB DNA binding ELISA of nuclear extracts from IT848 treated representative DLBCL and multiple myeloma cell lines showed decreased DNA binding of all five NF-κB subunits (although with cell type-specific variability; Fig. 1H). We next assessed the specificity of IT848 and found that inhibition of transcriptional activity was specific to NF-κB but spared unrelated transcription factors that were tested as controls (NFAT and Nrf2; Fig. 1I). Taken together, our findings identify IT848 as a potent and specific small molecule NF-κB inhibitor that decreases the affinity of NF-κB subunits to their DNA binding sites and disrupts downstream biological events.

NF-κB is known to be constitutively active in all multiple myeloma cells (5, 6, 25). Indeed, NF-κB DNA binding analysis of two multiple myeloma cell lines (MM.1S and U266) confirmed constitutive activity of all five NF-κB subunits (Supplementary Fig. S3). To assess the anti-myeloma activity of IT848 we treated MM.1S, U266, and NCI-H929 cells with increasing doses of the compound. This resulted in a dose-dependent decrease in cell numbers over a 72-hour period as measured by luciferase assay (Fig. 2A) and direct cell counting (Fig. 2B). Importantly, PBMCs of several healthy control subjects were not negatively affected by IT848 treatment except for a 10 percent decrease in metabolic viability in the presence of the highest tested concentration (6 μmol/L; Fig. 2C). To further evaluate the mechanism of action associated with the cytotoxic effects of IT848, we examined its impact on cell-cycle phase distribution after 24 hours of treatment. The percentages of IT848-treated cells in S phase were increased while the frequency of cells in G₂–M phase was lowered in a dose-dependent manner (Fig. 2D). This suggests that IT848 effectively affects cell-cycle checkpoints leading to S phase arrest and inhibition of cell division, which was confirmed by concordant decreased levels of Ki-67 expression, a nuclear protein present in proliferating cells (Fig. 2E). Again, normal PBMCs were insensitive to the anti-proliferative effect of IT848 (Fig. 2E). Furthermore, Annexin-V and 7-AAD staining revealed that IT848 induced a shift from early apoptosis (Annexin V+, 7-AAD−) to multiple myeloma cell death (Annexin V+, 7-AAD+) after 24 hours of treatment (Fig. 2F). Therefore, the IT848-induced decrease in cell numbers is likely due to a dual effect, including inhibition of proliferation and induction of apoptosis. IT848 treatment resulted in a dose-dependent increase in caspase activity in multiple myeloma cells (using a kit detecting activity of caspase 1, 3, 4, 5, 6, 7, 8 and 9; Fig. 2G). Furthermore, IT848 treatment enhanced nuclear translocation of apoptosis-inducing factor (AIF), a factor involved in a caspase-independent pathway of apoptosis (Fig. 2H; refs. 26–28). AIF is a mitochondrial proapoptotic factor that is released from mitochondria upon decrease of the mitochondrial membrane potential and subsequent increased permeability of the mitochondrial membrane (26, 27). We found that while low-dose IT848 treatment (2 and 4 μmol/L) did not alter the mitochondrial membrane potential of multiple myeloma cells, higher doses triggered a loss of the mitochondrial membrane potential (Fig. 2I), consistent with bioenergetic stress and release of proapoptotic factors such as AIF.

In contrast to antibody-based targeted therapy, chemical approaches using small molecules are expected to be less specific and off-target effects are common. Therefore, to broadly assess the effects of IT848 on cellular processes in multiple myeloma cells we performed RNA-seq of U266 and MM.1S cells treated with IT848 for 6 hours (Fig. 3A and B). This timepoint was chosen because significant cell death has not occurred yet and it therefore allows to assess the impact of IT848 treatment on the transcriptome of multiple myeloma cells. As expected, IT848 treatment of MM.1S cells resulted in downregulation of a variety of NF-κB target genes (Supplementary Table S1). However, we also found highly significant upregulations of reactive oxygen species (ROS) and apoptosis pathway genes, including heme oxygenase 1 (HMOX1) and oxidative stress-induced growth inhibitor 1 (OSGIN1), following exposure to IT848 (Fig. 3A and B). GSEA revealed that oxidative stress and apoptosis signatures were among the most significantly enriched gene sets in myeloma cells treated with IT848 (Fig. 3C; Supplementary Fig. S4). We validated RNA-seq findings regarding HMOX1 and OSGIN1 using RT-qPCR (Fig. 4A and B). Remarkably, we showed that the effect of IT848 on oxidative stress was selective for multiple myeloma cells while only minimal changes were observed in normal PBMCs (Fig. 4A and B). Specifically, IT848 treatment induced an increase of cellular superoxide (Fig. 4C) but not of hydrogen peroxide levels. Glutathione (GSH) is one of the major ROS-scavenger systems protecting cells from oxidative stress. We therefore compared GSH levels in MM.1S and U266 cells with and without IT848 treatment and found up to 70% depletion of the GSH pool with increasing drug concentrations (Fig. 4D). Control cells (splenocytes and NIH-3T3 cells) showed a negligible inhibition pattern, reinforcing our previous observation that myeloma cells are selectively sensitive to oxidative stress induction by IT848. Repletion of GSH partially reversed the anti-myeloma effect of IT848 in assays monitoring cell growth (Fig. 4E) and apoptosis (Supplementary Fig. S5), validating GSH depletion in cancer cells as a critical component of the mechanism of action of our drug. For in vivo confirmation of its pro-oxidant properties, we tested IT848-mediated ROS induction in NGS mice inoculated with MM.1S cells. Administration of the light emitting molecule coelenterazine resulted in enhanced chemiluminescent detection of superoxide anion (29) in the treatment group, suggesting that IT848 treatment can achieve in vivo modulation of the cellular redox homeostasis (Fig. 4F).

Next, we investigated the PK of IT848 by analyzing its whole blood or plasma levels following oral, intraperitoneal, or intravenous administration (Supplementary Fig. S6). We found that the maximum plasma concentration (c_max) and half-life (t_1/2) of IT848 following all three routes of administration was sufficient to achieve sustained plasma concentrations equivalent to the 4 to 6 μmol/L range, the therapeutic window associated with in vitro efficacy (see Fig. 2A and B). In addition, toxicology studies demonstrated the absence of organ toxicities during a 12-day course of IT848 treatment.

For in vivo efficacy testing of IT848 we first utilized a xenograft model of multiple myeloma and included the proteasome inhibitor bortezomib (30, 31). We chose bortezomib as a reference drug because it is a well-established anti-myeloma agent with NF-κB inhibitory properties but a different mechanism of action than IT848, hypothesizing that combination therapy should be safe and may producde additive effects. Mice were treated with either vehicle (control), bortezomib monotherapy, IT848 monotherapy, or the combination of the two. Monitoring disease progression by in vivo BLI, we observed significant efficacy with both bortezomib (P = 0.008) and IT848 monotherapy (P < 0.001; Fig. 5A and B). Notably, IT848 showed superior tumor growth inhibition compared to bortezomib (P = 0.032), and combination treatment resulted in improved efficacy when compared with either monotherapy (P < 0.001, P = 0.029). RNA-seq comparing MM.1S cells treated with bortezomib alone versus bortezomib plus IT848 (Supplementary Fig. S7) revealed a more significant downregulation of seven NF-κB related genes with combination treatment, including the NF-κB essential modulator (NEMO; P = 0.02), which is a validated target for molecular cancer therapy (32–34).

However, frequent intraperitoneal administration, which is a clincially irrelevant drug devivery route but commonly used in preclinical animal models, of IT848 for more than two weeks was associated with considerable toxicity (such as development of ascites and diarrhea) and concordant treatment-associated mortality in the range of 20 to 30 percent (Fig. 5A; Supplementary Fig. S8). Analysis of cause-specific survival revealed that despite this mouse model-specific drawback, the compound still mediated therapeutically beneficial in vivo efficacy resulting in significantly improved disease-specific survival (Fig. 5C). These observations suggest the need for the development of more clinically relevant better tolerated treatment protocols based on intravenous or oral delivery routes. Importantly, we were recently able to demonstrate that less frequent dosing of intravenously delivered IT848 was efficacious in the absence of toxicity (Supplementary Fig. S9), and additional studies with the goal to refine and optimize our protocols are currently underway.

Encouraged by the effectiveness of IT848 against multiple myeloma cell lines both in vitro and in vivo we sought to assess the response of primary multiple myeloma cells to IT848. We therefore performed apoptosis and proliferation assays with multiple myeloma samples derived from 2 patients with newly diagnosed multiple myeloma and 1 patient with highly aggressive advanced disease (plasma cell leukemia). These assays confirmed that IT848 induced cell death (Fig. 5D) and inhibited proliferation in a dose-dependent manner (Fig. 5E) not just in multiple myeloma cell lines but also primary multiple myeloma cells.

Finally, given the known immunomodulatory effects of NF-κB inhibition, we investigated whether IT848 treatment could be exploited to maximize immunotherapeutic approaches in multiple myeloma. We previously demonstrated that small molecules inhibiting NF-κB DNA binding are active against alloimmunity (acute graft-versus-host disease) without compromising T cell–mediated protective immunity (21, 35). Furthermore, we recently discovered that our drug candidate IT848 was active against autoimmunity (rheumatoid arthritis model, Supplementary Fig. S10) and did not reduce the cytotoxic activity of CAR T cells (Supplementary Fig. S11). Because NF-κB regulates PD-L1 expression in B-cell lymphomas (8), we assessed PD-L1 levels in lymphoma and multiple myeloma cells treated with IT848 and found that IT848 treatment resulted in the downregulation of PD-L1 expression in both cell types (Supplementary Fig. S12), suggesting that IT848 might be a promising candidate for combination therapy with PD-1 blockade in B-cell malignancies. In addition to suppression of the PD-1/PD-L1 axis, NF-κB inhibition might enhance anti-myeloma immunity by modulating either myeloid-derived suppressor cells (MDSC) or regulatory T cells (Treg) in the bone marrow (BM) microenvironment in the setting of multiple myeloma (36–38), providing an additional rationale for combining IT848 with an immune checkpoint inhibitor strategy. To test our hypothesis we investigated the efficacy of IT848/anti–PD-1 combination both in vitro and in vivo in the immunocompetent mouse model 5TGM1 (16). In vitro coculture experiments of BM mononuclear cells and 5TGM1 cells in the presence and absence of IT848, anti–PD-1, or their combination, revealed that only combination treatment resulted in significant anti-myeloma activity (Fig. 6A). Furthermore, PD-1 expression was decreased on all T cells of the combination treatment group (Fig. 6B). While this decrease in PD-1 expression also included Tregs (Supplementary Fig. S13A), analysis of Ki-67 expression by PD-1–positive Tregs showed no evidence of Treg hyperproliferation (Supplementary Fig. S13A), a phenomenon that has been associated with cancer progression following anti–PD-1 therapy (39). Combination treatment also resulted in increased CD25 expression by cytotoxic T lymphocytes (CTL; Fig. 6B) and an increase in the frequency of activated CTLs (CD25⁺IFNγ⁺; Fig. 6B). We also analyzed immunosuppressive, Treg-promoting growth factors and cytokines such as TGFβ and IL10 (40). However, intracellular cytokine analysis of M-MDSCs and PMN-MDSCs showed overall no significant alterations in TGFβ and IL10 expression in any of the tested conditions (Supplementary Fig. S13B). We next compared the same conditions (control, IT848 monotherapy, anti–PD-1 monotherapy, and combination therapy) in the 5TGM1 model in vivo. We found that a 14-day treatment course of IT848/anti–PD-1 reduced multiple myeloma progression and improved progression free survival (Fig. 6C and D). Amongst immunosuppressive cell populations in the BM of multiple myeloma bearing mice, the number of Tregs (but not of MDSCs) was significantly decreased in mice receiving IT848/anti–PD-1 combination treatment (Fig. 6E, Supplementary Fig. S14), and CD25 expression of Tregs was lowest in Tregs of the combination therapy group (Fig. 6E), suggestive of decreased Treg-mediated immunosuppression of the TME. Collectively, our findings suggest that the observed therapeutic effect of IT848/anti–PD-1 combination therapy was at least partially due to modulation of the immunosuppressive BM microenvironment.

Even though constitutive NF-κB activity is crucial to the survival and progression of multiple myeloma cells and plays a role in the development of resistance to cancer therapeutics, targeting NF-κB safely remains a challenging balancing act. In addition, the crosstalk between myeloma cells and the immune components of the TME within the BM further complicates the therapeutic design (41). As a result, even though hundreds of NF-κB inhibitor compounds have been designed over the years, to date there are no approved agents that directly inhibit NF-κB transactivation (42). However, multiple indirect strategies to target NF-κB, including agents that were not originally conceived as NF-κB inhibitors, have yielded some significant advances. Examples include immunomodulatory drugs (IMiD) such as lenalidomide that suppresses IκB kinase (IKK) activity (via TNFα, IL1β inhibition; ref. 43), and proteasome inhibitors such as bortezomib and carfilzomib that target IκBα degradation (30). These classes of drugs have been used in multiple myeloma and B-cell lymphoma but lack specificity, have off-target effects, and dose-limiting toxicities (31, 44). Inhibitors of a central regulator of NF-κB activation, the IKK complex (45), on the other hand, are highly NF-κB–specific, but their efficacy in clinical trials has been underwhelming (46). As far as pharmaceutical inhibition of NF-κB DNA binding is concerned, a limited number of molecules (DHMEQ, SEMBL, PBS-1086) derived from the natural compound epoxyquinomicin C have been researched in the past, but the poor bioavailability and PK restricted their use and potential for clinical translation (47–49). On the other hand, our synthetic compound IT848 is characterized by drug-like physicochemical properties. IT848 inhibits NF-κB transcriptional activity by reducing the ability of NF-κB subunits that migrated to the nucleus to bind to DNA, thereby interfering with both canonical and noncanonical NF-κB signaling. Importantly, during healthy steady state conditions, NF-κB is kept in an inactive state in the cytoplasm and as such, in the absence of NF-κB activating stimuli, healthy cells are not expected to be negatively affected by pharmaceuticals specifically preventing DNA binding of NF-κB. Although small molecules alone are unlikely to induce durable responses in aggressive cancers such as multiple myeloma, we demonstrated significant in vivo efficacy of IT848 monotherapy. The above-mentioned adverse events following prolonged courses of clinically irrelevant intraperitoneal administration of IT848 in mice (e.g., diarrhea, ascites) do not occur with intravenous or oral administrations; both administration routes are viable options, given the favorable PK profile of IT848 and its efficacy in recently used both oral and intravenous regimens in mouse models of T-cell lymphoma (Supplementary Fig. S9) and rheumatoid arthritis (Supplementary Fig. S10).

We found an additional mechanism of action of IT848 that exploits the heightened ROS production in cancer cells, a consequence of redox homeostasis imbalance, oncogenic stimulation, increased metabolic activity, and mitochondrial malfunction (50, 51). We observed a dose-dependent increase in superoxide levels and a decrease of GSH in multiple myeloma cell lines but not in healthy control cells where basal ROS levels are minimal. A previous study found a direct correlation between GSH depletion and ROS-mediated apoptosis of cancer cells (52). Notably, NF-κB activity is required for cellular GSH production (53), and GSH depletion mediated specifically by NF-κB inhibition has been proposed as a therapeutic strategy for cancer treatment (53). Our findings were further reiterated by RNA-seq and qPCR data supporting a transcriptional upregulation of oxidative stress signatures and genes involved in oxidative stress-induced apoptosis. Collectively, our data indicate that therapeutic manipulation by IT848 can preferentially kill myeloma cells via NF-κB/ROS-mediated mechanisms utilizing both caspase-dependent and independent apoptosis pathways. Induction of cancer cell–selective oxidative stress by IT848 is closely related to GSH depletion and inadequate upregulation of oxidative stress response genes, which can both be attributed to the NF-κB inhibitory effects of the molecule. This suggests that IT848 has the potential to be effective against a wide range of cancer cells and its clinical application may not have to be limited to cancer cell types that rely on constitutive NF-κB activity for their survival.

We showed that IT848 induces prominent anti-myeloma activity against cell lines, primary cells, and in a xenograft model, where combining IT848 with bortezomib enhanced antitumor efficacy. However, because tumor modeling in the absence of a functional immune system limits clinical relevance (especially when studying approaches that have the potential to alter immune responses), we additionally explored the effect of IT848 in combination with PD-1 blockade in an immunocompetent mouse model of multiple myeloma. It has been shown that malignant plasma cells are able to induce T-cell anergy via PD-L1/PD-1 interaction (54), and PD-L1 expression in multiple myeloma cells correlates with BCMA signaling, which activates the canonical NF-κB pathway (55). We found that single agent treatment with either anti–PD-1 checkpoint blockade or IT848 was not effective, but combination therapy significantly decreased infiltration of the BM by Tregs and multiple myeloma cells, and it improved survival. In vitro testing of this combination therapy confirmed that combination of direct NF-κB inhibition with immune checkpoint blockade induces a shift of the multiple myeloma microenvironment toward enhanced cytotoxicity. MDSC populations were not quantitatively affected by IT848 or anti–PD-1 treatment, and there was no evidence of a role of MDSCs in promoting Treg activity through cytokine release. However, we found that IT848/anti–PD-1 combination treatment resulted in significant downregulation of the IL2 receptor alpha chain (CD25), which is an NF-κB target gene (56), in Tregs, suggesting that decreased responsiveness to the Treg growth factor IL2 may be a contributing factor for the depletion of BM resident Tregs (57). In addition, IT848 likely suppresses thymic Treg development, given that NF-κB regulates FoxP3 expression (58). Finally, it is well established that immune checkpoint blockade can be associated with autoreactive T cell–mediated adverse events such as colitis or hepatitis. Our previous findings in GVHD (35) and rheumatoid arthritis models suggest that IT848 treatment might ameliorate autoimmune-related adverse events associated with immune checkpoint blockade (59) without compromising antitumor activity of T cells (21, 35), providing an additional rationale for the development of IT848 combination therapies with immune checkpoint blockade. Taken together, these cellular and molecular interactions suggest that while high-dose IT848 displays antitumor activity in the absence of a functional immune system, harnessing the immunomodulary effects of IT848 for cancer immunotherapy promises synergistic therapeutic effects at lower doses in the absence of toxicities. Studies designed to further validate the latter concept are currently underway.

In conclusion, our study shows that the small molecule IT848 is a novel and highly specific type of NF-κB inhibitor and cancer cell–selective oxidative stress inducer with significant anti-myeloma activity against both cell lines and primary cells. IT848 demonstrated in vivo efficacy as single agent and in combination therapies designed to overcome immunosuppression and drug resistance in the TME. IT848 has a favorable PK profile and we found no toxicities associated with clinically relevant administration routes. Our data therefore provide a strong preclinical rationale for the clinical development of IT848 in B-cell malignancies.

I. Colorado reports grants from IMS during the conduct of the study. R. Feinman reports grants from International Myeloma Society during the conduct of the study. D.S. Siegel reports other support from BMS, Takeda, GSK, Merck, Janssen, Amgen, Karyopharm, IMF, Celularity, Neximmune; and other support from Sanofi outside the submitted work. J.L. Zakrzewski reports grants from ImmuneTarget Inc., Leukemia & Lymphoma Society; and grants from International Myeloma Society during the conduct of the study. No disclosures were reported by the other authors.

M. Bariana: Data curation, formal analysis, validation, investigation, visualization, methodology, writing–original draft. E. Cassella: Data curation, formal analysis, methodology. J. Rateshwar: Data curation, formal analysis, methodology. S. Ouk: Resources, data curation, investigation. H.-C. Liou: Resources, data curation, investigation. C. Heller: Data curation, investigation. I. Colorado: Investigation. R. Feinman: Resources, supervision, investigation, methodology. A. Makhdoom: Data curation, investigation. D.S. Siegel: Resources, project administration. G. Heller: Formal analysis, methodology. A. Tuckett: Data curation, formal analysis, investigation. P. Mondello: Data curation, formal analysis, supervision, investigation, methodology, writing–review and editing. J.L. Zakrzewski: Conceptualization, resources, formal analysis, supervision, funding acquisition, validation, project administration, writing–review and editing.

This study was supported by a Sponsored Research Agreement from ImmuneTarget Inc. to J.L. Zakrzewski. J.L. Zakrzewski also received grant support from the NCI (NCI 1R37CA250661-01A1), the Leukemia and Lymphoma Society (grant # 6465-15), the American Society of Hematology, the International Myeloma Society, Hyundai Hope on Wheels, the HMH MSK Immunology Collaboration, and the HMH Foundation/Tackle Kids Cancer. IT848 was provided by Immunetarget, Inc.

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