In This Issue

See article, p. 920.

Patients who present with metastatic osteosarcoma have poor prognosis. However, only a few genes have been implicated in osteosarcoma, and no genes have been associated with osteosarcoma metastasis. To identify risk variants associated with metastasis, Mirabello and colleagues analyzed 935 cases of osteosarcoma in a multistage genome-wide association study (GWAS) of osteosarcoma metastasis. This analysis identified two intronic SNPs at 9p24.1 in the nuclear factor I/B (NFIB) gene, which encodes a transcription factor that has not been previously implicated in osteosarcoma progression or metastasis. Both SNPs were highly associated with increased risk of metastasis at diagnosis, and the risk allele rs7034162 was associated with increased risk of metastasis across three ethnic groups, worse overall survival, and decreased NFIB expression. In vitro analyses revealed that the level of endogenous NFIB expression was inversely correlated with migration and invasion in osteosarcoma cell lines and that overexpression of NFIB in a cell line with low endogenous NFIB reduced soft-agar colony formation. The risk allele rs7034162 was also associated with decreased expression of IGFBP5, a known transcriptional target of NFIB that has been shown to inhibit growth and metastasis of osteosarcoma cells. In addition, analysis of osteosarcomas and metastases from a Sleeping Beauty transposon screen in mice identified inactivating transposon insertions in Nfib, which resulted in reduced expression of both Nfib and Igfbp5. These findings show that germline genetic variation in NFIB is associated with risk of osteosarcoma metastasis at diagnosis.

See article, p. 932.

The initial step of metastasis is tumor cell dissemination into the tumor vasculature, which has been well-characterized as morphologically abnormal and hyperpermeable. However, the mechanism underlying the spatiotemporal heterogeneity of tumor vasculature permeability has been difficult to elucidate, and consequently the interaction between tumor vasculature and tumor cells during dissemination has not been precisely described. Using intravital high-resolution two-photon microscopy (IVM), Harney and colleagues were able to study tumor dissemination of mouse models of breast and mammary carcinoma in the Tumor MicroEnvironment of Metastasis (TMEM), which is a micro-anatomical site comprised of tumor-associated macrophages (TAM) in direct contact with a tumor cell overexpressing the actin-binding protein mammalian-enabled (MENA) and an endothelial cell. Real-time IVM revealed that tumor cells and macrophages migrate to sites of transient vascular permeability in the TMEM where tumor cells then undergo transendothelial migration, and that tumor cell intravasation occurs concurrently with transient vascular permeability and is restricted to the TMEM. The TMEM of mouse mammary tumors is enriched for TIE2^hi/VEGFA^hi macrophages, and inhibition of VEGFA or ablation of TIE2^hi/VEGFA^hi macrophages reduces vascular permeability and tumor cell intravasation. Given that loss of vascular junction integrity in vasculature adjacent to TIE2^hi/VEGFA^hi macrophages was observed in both mouse mammary tumors and breast cancer patient samples, these results suggest not only that TIE2^hi/VEGFA^hi macrophages induce vascular permeability and tumor intravasation in TMEMs, but also that therapeutic targeting of TMEMs to suppress metastasis may be warranted.

See article, p. 944.

Upregulation of the receptor tyrosine kinases EGFR and HER2 has been implicated in acquired resistance to endocrine therapies such as tamoxifen in estrogen receptor (ER)–positive breast cancer. Recent studies have suggested that the homeobox protein HOXB7 contributes to tamoxifen resistance via activation of ER target genes, but the role of HOXB7 in the regulation of ER transcriptional activity is unknown. Jin and colleagues found that HOXB7 bound to ER binding sites in chromatin and promoted the expression of ER target genes, including MYC and HER2, via direct interaction with ER and recruitment of ER coactivator proteins in tamoxifen-resistant breast cancer cells. Analysis of upstream pathways revealed that hyperphosphorylation and stabilization of MYC by EGFR/HER2 resulted in MYC-mediated transcriptional repression of miR-196a and subsequent HOXB7 upregulation in tamoxifen-resistant cells. Depletion of HOXB7 or inhibition of MYC in tamoxifen-resistant cells reduced expression of HER2 and ER target genes, restored tamoxifen sensitivity, and synergized with HER2-targeted therapies to inhibit ER-positive xenograft growth. Consistent with these findings, restoration of miR-196a expression in tamoxifen-resistant cells attenuated tamoxifen resistance and induced tumor regression in combination with tamoxifen. Furthermore, coexpression of HOXB7, HER2, and MYC was prognostic of poor overall and relapse-free survival in patients with ER-positive breast cancer who received endocrine therapy. These findings identify HOXB7 as an ER cofactor and suggest that targeting the MYC–HOXB7–HER2 pathway may limit resistance to endocrine therapy in ER-positive breast cancer.

See article, p. 960.

Long-term EGFR tyrosine kinase inhibitor (TKI) efficacy is hindered by acquired resistance mechanisms, including secondary mutation at EGFR^T790M and EGFR-independent activation of ERK1/2 in EGFR-mutant non–small cell lung cancer (NSCLC). To prevent the development of resistance, Tricker, Xu, and colleagues investigated the efficacy of a cotreatment strategy involving the mutant EGFR–selective inhibitor WZ4002 and the MEK inhibitor trametinib. In contrast to WZ4002 treatment alone, the combination of WZ4002 and trametinib reduced ERK1/2 reactivation and delayed the emergence of acquired resistance in TKI-naïve cells and cells that had developed TKI resistance specifically via the EGFR^T790M mutation. WZ4002/trametinib–sensitive cells exhibited sustained EGFR signaling inhibition and elevated caspase 3 apoptotic activity; however, resistant cells reactivated AKT and S6 despite EGFR and ERK1/2 inhibition. Similar to in vitro results, the WZ4002/trametinib combination was significantly more effective than WZ4002 alone in preventing tumor regrowth after initial regression in xenograft models and a genetically engineered mouse model of EGFR^T790M-mutant NSCLC. Although EGFR and ERK inhibition was maintained in the majority of resistant tumor nodules, both AKT and S6 were frequently reactivated, and the addition of an mTOR inhibitor to WZ4002 and trametinib suppressed the growth of WZ4002/trametinib–resistant cell lines in vitro and tumors in vivo. These results highlight the potential clinical utility of initial cotargeting of EGFR and MEK to prolong EGFR and ERK1/2 inhibition, increase apoptosis, and ultimately prevent the emergence of acquired resistance in EGFR-mutant NSCLC tumors.

See article, p. 972.

The genomic landscape of multiple myeloma is complex, consisting of aberrant chromosomal karyotypes and DNA copy-number alterations. Although the source of genomic instability remains unclear, upregulation of oncogenes such as MYC has been suggested to promote replicative or oxidative stress and to activate the DNA damage response. In an effort to dissect the molecular mechanisms that drive DNA damage in multiple myeloma, Cottini and colleagues showed that a subset of multiple myeloma cell lines displayed increased expression of DNA replication and cell-cycle regulators, as well as markers of replicative stress. In line with this finding, a subset of patients with multiple myeloma exhibited upregulation of a gene signature previously linked with genomic instability, which was associated with poor prognosis, and harbored increased levels of MYC. Mechanistically, increased MYC expression activated replicative stress–associated DNA damage and promoted the production of reactive oxygen species (ROS) as a result of mitochondrial deregulation. MYC-overexpressing cells required ATR activation for survival under replicative stress, and augmentation of DNA damage, via either pharmacologic ATR inhibition or treatment with the ROS-inducing molecule piperlongumine, induced apoptosis in multiple myeloma cells with ongoing DNA damage. Furthermore, the combination of ATR inhibition and piperlongumine led to synergistic cytotoxic effects, indicative of a synthetic lethal effect. Together, these data suggest that MYC-driven DNA damage may be therapeutically exploited in a subset of patients with multiple myeloma characterized by inherent genomic instability.

See article, p. 988.

Acute myeloid leukemia (AML) derives from malignant leukemia stem and progenitor cells (LSC) and can be divided into molecularly distinct subtypes. LSCs have also been hypothesized to drive relapse due to their protective microenvironment and quiescent nature. However, more recent studies suggest that subtype-specific genetic aberrations may drive chemotherapy resistance. In order to better understand the molecular factors that mediate therapy response in AML, Behbehani and colleagues used mass cytometry–based high-dimensional functional profiling to measure the immunophenotypic, cell-cycle, and intracellular signaling properties of patient-derived AML cells. This analysis revealed that patients with AML harboring specific genetic aberrations, such as core-binding factor mutations or adverse-risk karyotypes, were characterized by a higher prevalence of immature immunophenotypes, and identified AML subtype–specific immunophenotypic aberrancies in surface marker expression in hematopoietic stem and progenitor cells (HSPC). In addition, cell-cycle labeling of bone-marrow aspirates highlighted the overall reduced proliferative rate of AML HSPCs compared with cells from healthy individuals, and demonstrated that chemotherapy-sensitive AML subtypes had LSCs with a higher proliferative fraction. Analysis of intracellular signaling pathways revealed increased ERK signaling across AML subtypes and subtype-specific activation of certain pathways. Furthermore, mass cytometry profiling showed that hydroxyurea chemotherapy did not alter the proliferative fraction of most myeloid immunophenotypic populations, suggesting that current in vitro cell models may not accurately reflect in vivo responses. Together, these data provide evidence that mass cytometry–based profiling may stratify patients based on AML subtype and predict therapeutic response.

Note: In This Issue is written by Cancer Discovery Science Writers. Readers are encouraged to consult the original articles for full details.