Combinatorial strategies to target RAS-driven cancers.

Although RAS was formerly considered undruggable, various agents that inhibit RAS or specific RAS oncoproteins have now been developed. Indeed, the importance of directly targeting RAS has recently been illustrated by the clinical success of mutant-selective KRAS inhibitors. Nevertheless, responses to these agents are typically incomplete and restricted to a subset of patients, highlighting the need to develop more effective treatments, which will likely require a combinatorial approach. Vertical strategies that target multiple nodes within the RAS pathway to achieve deeper suppression are being investigated and have precedence in other contexts. However, alternative strategies that co-target RAS and other therapeutic vulnerabilities have been identified, which may mitigate the requirement for profound pathway suppression. Regardless, the efficacy of any given approach will likely be dictated by genetic, epigenetic and tumour-specific variables. Here we discuss various combinatorial strategies to treat KRAS-driven cancers, highlighting mechanistic concepts that may extend to tumours harbouring other RAS mutations. Although many promising combinations have been identified, clinical responses will ultimately depend on whether a therapeutic window can be achieved and our ability to prospectively select responsive patients. Therefore, we must continue to develop and understand biologically diverse strategies to maximize our likelihood of success.

Cotargeting a MYC/eIF4A-survival axis improves the efficacy of KRAS inhibitors in lung cancer.

Despite the success of KRAS G12C inhibitors in non-small cell lung cancer (NSCLC), more effective treatments are needed. One preclinical strategy has been to cotarget RAS and mTOR pathways; however, toxicity due to broad mTOR inhibition has limited its utility. Therefore, we sought to develop a more refined means of targeting cap-dependent translation and identifying the most therapeutically important eukaryotic initiation factor 4F complex-translated (eIF4F-translated) targets. Here, we show that an eIF4A inhibitor, which targets a component of eIF4F, dramatically enhances the effects of KRAS G12C inhibitors in NSCLCs and together these agents induce potent tumor regression in vivo. By screening a broad panel of eIF4F targets, we show that this cooperativity is driven by effects on BCL-2 family proteins. Moreover, because multiple BCL-2 family members are concomitantly suppressed, these agents are broadly efficacious in NSCLCs, irrespective of their dependency on MCL1, BCL-xL, or BCL-2, which is known to be heterogeneous. Finally, we show that MYC overexpression confers sensitivity to this combination because it creates a dependency on eIF4A for BCL-2 family protein expression. Together, these studies identify a promising therapeutic strategy for KRAS-mutant NSCLCs, demonstrate that BCL-2 proteins are the key mediators of the therapeutic response in this tumor type, and uncover a predictive biomarker of sensitivity.

USP9X mediates an acute adaptive response to MAPK suppression in pancreatic cancer but creates multiple actionable therapeutic vulnerabilities.

Pancreatic ductal adenocarcinomas (PDACs) frequently harbor KRAS mutations. Although MEK inhibitors represent a plausible therapeutic option, most PDACs are innately resistant to these agents. Here, we identify a critical adaptive response that mediates resistance. Specifically, we show that MEK inhibitors upregulate the anti-apoptotic protein Mcl-1 by triggering an association with its deubiquitinase, USP9X, resulting in acute Mcl-1 stabilization and protection from apoptosis. Notably, these findings contrast the canonical positive regulation of Mcl-1 by RAS/ERK. We further show that Mcl-1 inhibitors and cyclin-dependent kinase (CDK) inhibitors, which suppress Mcl-1 transcription, prevent this protective response and induce tumor regression when combined with MEK inhibitors. Finally, we identify USP9X as an additional potential therapeutic target. Together, these studies (1) demonstrate that USP9X regulates a critical mechanism of resistance in PDAC, (2) reveal an unexpected mechanism of Mcl-1 regulation in response to RAS pathway suppression, and (3) provide multiple distinct promising therapeutic strategies for this deadly malignancy.

Combating castration-resistant prostate cancer by co-targeting the epigenetic regulators EZH2 and HDAC.

While screening and early detection have reduced mortality from prostate cancer, castration-resistant disease (CRPC) is still incurable. Here, we report that combined EZH2/HDAC inhibitors potently kill CRPCs and cause dramatic tumor regression in aggressive human and mouse CRPC models. Notably, EZH2 and HDAC both transmit transcriptional repressive signals: regulating histone H3 methylation and histone deacetylation, respectively. Accordingly, we show that suppression of both EZH2 and HDAC are required to derepress/induce a subset of EZH2 targets, by promoting the sequential demethylation and acetylation of histone H3. Moreover, we find that the induction of one of these targets, ATF3, which is a broad stress response gene, is critical for the therapeutic response. Importantly, in human tumors, low ATF3 levels are associated with decreased survival. Moreover, EZH2- and ATF3-mediated transcriptional programs inversely correlate and are most highly/lowly expressed in advanced disease. Together, these studies identify a promising therapeutic strategy for CRPC and suggest that these two major epigenetic regulators buffer prostate cancers from a lethal response to cellular stresses, thereby conferring a tractable therapeutic vulnerability.

DAB2IP Is a Bifunctional Tumor Suppressor That Regulates Wild-Type RAS and Inflammatory Cascades in KRAS Mutant Colon Cancer.

The DAB2IP tumor suppressor encodes a RAS GTPase-activating protein. Accordingly, DAB2IP has been shown to be mutated or suppressed in tumor types that typically lack RAS mutations. However, here we report that DAB2IP is mutated or selectively silenced in the vast majority of KRAS and BRAF mutant colorectal cancers. In this setting, DAB2IP loss promoted tumor development by activating wild-type H- and N-RAS proteins, which was surprisingly required to achieve robust activation of RAS effector pathways in KRAS-mutant tumors. DAB2IP loss also triggered production of inflammatory mediators and the recruitment of protumorigenic macrophages in vivo. Importantly, tumor growth was suppressed by depleting macrophages or inhibiting cytokine/inflammatory mediator expression with a JAK/TBK1 inhibitor. In human tumors, DAB2IP was lost at early stages of tumor development, and its depletion was associated with an enrichment of macrophage and inflammatory signatures. Together, these findings demonstrate that DAB2IP restrains the activation of the RAS pathway and inflammatory cascades in the colon and that its loss represents a common and unappreciated mechanism for amplifying these two critical oncogenic signals in colorectal cancer. SIGNIFICANCE: DAB2IP is lost in early-stage tumors, which amplifies RAS signaling, triggers inflammatory mediators, and recruits macrophages in KRAS-mutant colon cancers.

Imaging Mass Spectrometry Reveals Tumor Metabolic Heterogeneity.

Malignant tumors exhibit high degrees of genomic heterogeneity at the cellular level, leading to the view that subpopulations of tumor cells drive growth and treatment resistance. To examine the degree to which tumors also exhibit metabolic heterogeneity at the level of individual cells, we employed multi-isotope imaging mass spectrometry (MIMS) to quantify utilization of stable isotopes of glucose and glutamine along with a label for cell division. Mouse models of melanoma and malignant peripheral nerve sheath tumors (MPNSTs) exhibited striking heterogeneity of substrate utilization, evident in both proliferating and non-proliferating cells. We identified a correlation between metabolic heterogeneity, proliferation, and therapeutic resistance. Heterogeneity in metabolic substrate usage as revealed by incorporation of glucose and glutamine tracers is thus a marker for tumor proliferation. Collectively, our data demonstrate that MIMS provides a powerful tool with which to dissect metabolic functions of individual cells within the native tumor environment.

A Deregulated HOX Gene Axis Confers an Epigenetic Vulnerability in KRAS-Mutant Lung Cancers.

While KRAS mutations are common in non-small cell lung cancer (NSCLC), effective treatments are lacking. Here, we report that half of KRAS-mutant NSCLCs aberrantly express the homeobox protein HOXC10, largely due to unappreciated defects in PRC2, which confers sensitivity to combined BET/MEK inhibitors in xenograft and PDX models. Efficacy of the combination is dependent on suppression of HOXC10 by BET inhibitors. We further show that HOXC10 regulates the expression of pre-replication complex (pre-RC) proteins in sensitive tumors. Accordingly, BET/MEK inhibitors suppress pre-RC proteins in cycling cells, triggering stalled replication, DNA damage, and death. These studies reveal a promising therapeutic strategy for KRAS-mutant NSCLCs, identify a predictive biomarker of response, and define a subset of NSCLCs with a targetable epigenetic vulnerability.

IL1α Antagonizes IL1ß and Promotes Adaptive Immune Rejection of Malignant Tumors.

We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1ß-/-, IL1α-/-, and IL1R1-/- mice. Tumors grew progressively in IL1R-/- and IL1α-/- mice but were often absent in IL1ß-/- mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1ß prevented tumor growth in wild-type (WT) mice but not in IL1R1-/- or IL1α-/- mice. Antibodies to IL1α promoted tumor growth in IL1ß-/- mice and reversed the tumor-suppressive effect of anti-IL1ß in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1ß-/- tumor suppressive effect, as did crossing IL1ß-/- mice to SCID or Rag1-/- mice. Finally, blockade of IL1ß synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1ß promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell-mediated antitumor immunity.

Targeting Refractory Sarcomas and Malignant Peripheral Nerve Sheath Tumors in a Phase I/II Study of Sirolimus in Combination with Ganetespib (SARC023).

PURPOSE: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas. Combining Hsp90 inhibitors to enhance endoplasmic reticulum stress with mTOR inhibition results in dramatic MPNST shrinkage in a genetically engineered MPNST mouse model. Ganetespib is an injectable potent small molecule inhibitor of Hsp90. Sirolimus is an oral mTOR inhibitor. We sought to determine the safety, tolerability, and recommended dose of ganetespib and sirolimus in patients with refractory sarcomas and assess clinical benefits in patients with unresectable/refractory MPNSTs. Patients and Methods. In this multi-institutional, open-label, phase 1/2 study of ganetespib and sirolimus, patients ≥16 years with histologically confirmed refractory sarcoma (phase 1) or MPNST (phase 2) were eligible. A conventional 3 + 3 dose escalation design was used for phase 1. Pharmacokinetic and pharmacodynamic measures were evaluated. Primary objectives of phase 2 were to determine the clinical benefit rate (CBR) of this combination in MPNSTs. Patient-reported outcomes assessed pain. RESULTS: Twenty patients were enrolled (10 per phase). Toxicities were manageable; most frequent non-DLTs were diarrhea, elevated liver transaminases, and fatigue. The recommended dose of ganetespib was 200 mg/m2 intravenously on days 1, 8, and 15 with sirolimus 4 mg orally once daily with day 1 loading dose of 12 mg. In phase 1, one patient with leiomyosarcoma achieved a sustained partial response. In phase 2, no responses were observed. The median number of cycles treated was 2 (1-4). Patients did not meet the criteria for clinical benefit as defined per protocol. Pain ratings decreased or were stable. CONCLUSION: Despite promising preclinical rationale and tolerability of the combination therapy, no responses were observed, and the study did not meet parameters for further evaluation in MPNSTs. This trial was registered with (NCT02008877).

Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer.

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

Targeting Sporadic and Neurofibromatosis Type 1 (NF1) Related Refractory Malignant Peripheral Nerve Sheath Tumors (MPNST) in a Phase II Study of Everolimus in Combination with Bevacizumab (SARC016).

PURPOSE: There are no known effective medical treatments for refractory MPNST. Inactivation of the NF1 tumor suppressor in MPNST results in upregulation of mTOR (mammalian target of rapamycin) signaling and angiogenesis, which contributes to disease progression. We conducted a phase II study for patients (pts) with refractory MPNST combining everolimus (10 mg PO once daily) with bevacizumab (10 mg/kg IV every 2 weeks) to determine the clinical benefit rate (CBR) (complete response, partial response (PR), or stable disease (SD) ≥ 4 months). PATIENTS AND METHODS: Patients ≥18 years old with chemotherapy refractory sporadic or NF1 MPNST were eligible. Tumor response was assessed after every 2 cycles (the WHO criteria). A two-stage design targeting a 25% CBR was used: if ≥ 1/15 pts in stage 1 responded, enrollment would be expanded by 10 pts, and if ≥ 4/25 patients had clinical benefit, the combination would be considered active. RESULTS: Twenty-five pts, 17 with NF1 and 8 with sporadic MPNST, enrolled. One of 15 pts in stage 1 had clinical benefit. Of 10 additional pts enrolled, 2 had clinical benefit. The median number of completed cycles was 3 (range 1-16). Adverse events were similar to those known for this combination. CONCLUSION: With a CBR of 12% (3/25), the combination of everolimus and bevacizumab did not reach the study's target response rate and is not considered active in refractory MPNST.

MAPK Pathway Suppression Unmasks Latent DNA Repair Defects and Confers a Chemical Synthetic Vulnerability in BRAF-, NRAS-, and NF1-Mutant Melanomas.

Although the majority of BRAF-mutant melanomas respond to BRAF/MEK inhibitors, these agents are not typically curative. Moreover, they are largely ineffective in NRAS- and NF1-mutant tumors. Here we report that genetic and chemical suppression of HDAC3 potently cooperates with MAPK pathway inhibitors in all three RAS pathway-driven tumors. Specifically, we show that entinostat dramatically enhances tumor regression when combined with BRAF/MEK inhibitors, in both models that are sensitive or relatively resistant to these agents. Interestingly, MGMT expression predicts responsiveness and marks tumors with latent defects in DNA repair. BRAF/MEK inhibitors enhance these defects by suppressing homologous recombination genes, inducing a BRCA-like state; however, addition of entinostat triggers the concomitant suppression of nonhomologous end-joining genes, resulting in a chemical synthetic lethality caused by excessive DNA damage. Together, these studies identify melanomas with latent DNA repair defects, describe a promising drug combination that capitalizes on these defects, and reveal a tractable therapeutic biomarker. SIGNIFICANCE: BRAF/MEK inhibitors are not typically curative in BRAF-mutant melanomas and are ineffective in NRAS- and NF1-mutant tumors. We show that HDAC inhibitors dramatically enhance the efficacy of BRAF/MEK inhibitors in sensitive and insensitive RAS pathway-driven melanomas by coordinately suppressing two DNA repair pathways, and identify a clinical biomarker that predicts responsiveness.See related commentary by Lombard et al., p. 469.This article is highlighted in the In This Issue feature, p. 453.

Genetic modifiers of the BRD4-NUT dependency of NUT midline carcinoma uncovers a synergism between BETis and CDK4/6is.

Bromodomain and extraterminal (BET) domain inhibitors (BETis) show efficacy on NUT midline carcinoma (NMC). However, not all NMC patients respond, and responders eventually develop resistance and relapse. Using CRISPR and ORF expression screens, we systematically examined the ability of cancer drivers to mediate resistance of NMC to BETis and uncovered six general classes/pathways mediating resistance. Among these, we showed that RRAS2 attenuated the effect of JQ1 in part by sustaining ERK pathway function during BRD4 inhibition. Furthermore, overexpression of Kruppel-like factor 4 (KLF4), mediated BETi resistance in NMC cells through restoration of the E2F and MYC gene expression program. Finally, we found that expression of cyclin D1 or an oncogenic cyclin D3 mutant or RB1 loss protected NMC cells from BETi-induced cell cycle arrest. Consistent with these findings, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors showed synergistic effects with BETis on NMC in vitro as well as in vivo, thereby establishing a potential two-drug therapy for NMC.

Cancer Cells Co-opt the Neuronal Redox-Sensing Channel TRPA1 to Promote Oxidative-Stress Tolerance.

Cancer cell survival is dependent on oxidative-stress defenses against reactive oxygen species (ROS) that accumulate during tumorigenesis. Here, we show a non-canonical oxidative-stress defense mechanism through TRPA1, a neuronal redox-sensing Ca2+-influx channel. In TRPA1-enriched breast and lung cancer spheroids, TRPA1 is critical for survival of inner cells that exhibit ROS accumulation. Moreover, TRPA1 promotes resistance to ROS-producing chemotherapies, and TRPA1 inhibition suppresses xenograft tumor growth and enhances chemosensitivity. TRPA1 does not affect redox status but upregulates Ca2+-dependent anti-apoptotic pathways. NRF2, an oxidant-defense transcription factor, directly controls TRPA1 expression, thus providing an orthogonal mechanism for protection against oxidative stress together with canonical ROS-neutralizing mechanisms. These findings reveal an oxidative-stress defense program involving TRPA1 that could be exploited for targeted cancer therapies.

Melanoma Therapeutic Strategies that Select against Resistance by Exploiting MYC-Driven Evolutionary Convergence.

Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, suggesting that durable control of resistance will be a challenge. By combining statistical modeling of genomic data from matched pre-treatment and post-relapse patient tumors with functional interrogation of >20 in vitro and in vivo resistance models, we discovered that major pathways of resistance converge to activate the transcription factor, c-MYC (MYC). MYC expression and pathway gene signatures were suppressed following drug treatment, and then rebounded during progression. Critically, MYC activation was necessary and sufficient for resistance, and suppression of MYC activity using genetic approaches or BET bromodomain inhibition was sufficient to resensitize cells and delay BRAFi resistance. Finally, MYC-driven, BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, including SRC family and c-KIT tyrosine kinases, as well as glucose, glutamine, and serine metabolic pathways. These insights enable the design of combination therapies that select against resistance evolution.

mTOR and HDAC Inhibitors Converge on the TXNIP/Thioredoxin Pathway to Cause Catastrophic Oxidative Stress and Regression of RAS-Driven Tumors.

Although agents that inhibit specific oncogenic kinases have been successful in a subset of cancers, there are currently few treatment options for malignancies that lack a targetable oncogenic driver. Nevertheless, during tumor evolution cancers engage a variety of protective pathways, which may provide alternative actionable dependencies. Here, we identify a promising combination therapy that kills NF1-mutant tumors by triggering catastrophic oxidative stress. Specifically, we show that mTOR and HDAC inhibitors kill aggressive nervous system malignancies and shrink tumors in vivo by converging on the TXNIP/thioredoxin antioxidant pathway, through cooperative effects on chromatin and transcription. Accordingly, TXNIP triggers cell death by inhibiting thioredoxin and activating apoptosis signal-regulating kinase 1 (ASK1). Moreover, this drug combination also kills NF1-mutant and KRAS-mutant non-small cell lung cancers. Together, these studies identify a promising therapeutic combination for several currently untreatable malignancies and reveal a protective nodal point of convergence between these important epigenetic and oncogenic enzymes.Significance: There are no effective therapies for NF1- or RAS-mutant cancers. We show that combined mTOR/HDAC inhibitors kill these RAS-driven tumors by causing catastrophic oxidative stress. This study identifies a promising therapeutic combination and demonstrates that selective enhancement of oxidative stress may be more broadly exploited for developing cancer therapies. Cancer Discov; 7(12); 1450-63. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1355.

A Collaborative Model for Accelerating the Discovery and Translation of Cancer Therapies.

Preclinical studies using genetically engineered mouse models (GEMM) have the potential to expedite the development of effective new therapies; however, they are not routinely integrated into drug development pipelines. GEMMs may be particularly valuable for investigating treatments for less common cancers, which frequently lack alternative faithful models. Here, we describe a multicenter cooperative group that has successfully leveraged the expertise and resources from philanthropic foundations, academia, and industry to advance therapeutic discovery and translation using GEMMs as a preclinical platform. This effort, known as the Neurofibromatosis Preclinical Consortium (NFPC), was established to accelerate new treatments for tumors associated with neurofibromatosis type 1 (NF1). At its inception, there were no effective treatments for NF1 and few promising approaches on the horizon. Since 2008, participating laboratories have conducted 95 preclinical trials of 38 drugs or combinations through collaborations with 18 pharmaceutical companies. Importantly, these studies have identified 13 therapeutic targets, which have inspired 16 clinical trials. This review outlines the opportunities and challenges of building this type of consortium and highlights how it can accelerate clinical translation. We believe that this strategy of foundation-academic-industry partnering is generally applicable to many diseases and has the potential to markedly improve the success of therapeutic development. Cancer Res; 77(21); 5706-11. ©2017 AACR.

Loss of RasGAP Tumor Suppressors Underlies the Aggressive Nature of Luminal B Breast Cancers.

Luminal breast cancers are typically estrogen receptor-positive and generally have the best prognosis. However, a subset of luminal tumors, namely luminal B cancers, frequently metastasize and recur. Unfortunately, the causal events that drive their progression are unknown, and therefore it is difficult to identify individuals who are likely to relapse and should receive escalated treatment. Here, we identify a bifunctional RasGAP tumor suppressor whose expression is lost in almost 50% of luminal B tumors. Moreover, we show that two RasGAP genes are concomitantly suppressed in the most aggressive luminal malignancies. Importantly, these genes cooperatively regulate two major oncogenic pathways, RAS and NF-κB, through distinct domains, and when inactivated drive the metastasis of luminal tumors in vivo Finally, although the cooperative effects on RAS drive invasion, NF-κB activation triggers epithelial-to-mesenchymal transition and is required for metastasis. Collectively, these studies reveal important mechanistic insight into the pathogenesis of luminal B tumors and provide functionally relevant prognostic biomarkers that may guide treatment decisions. SIGNIFICANCE: The lack of insight into mechanisms that underlie the aggressive behavior of luminal B breast cancers impairs treatment decisions and therapeutic advances. Here, we show that two RasGAP tumor suppressors are concomitantly suppressed in aggressive luminal B tumors and demonstrate that they drive metastasis by activating RAS and NF-κB. Cancer Discov; 7(2); 202-17. ©2016 AACR.See related commentary by Sears and Gray, p. 131This article is highlighted in the In This Issue feature, p. 115.