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Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK–MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K–AKT–mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor–based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC. Significance: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.

All supplemental data with figure legends

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF–MEK–ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF–MEK–ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.

Structure factors and crystal information

Supplementary Table from Concurrent Inhibition of ERK and Farnesyltransferase Suppresses the Growth of HRAS Mutant Head and Neck Squamous Cell Carcinoma

Supplementary Data from Concurrent Inhibition of ERK and Farnesyltransferase Suppresses the Growth of HRAS Mutant Head and Neck Squamous Cell Carcinoma

Supplementary Methods

Supplementary figures and legends

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF–MEK–ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF–MEK–ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.

Figure S1 page1

Supplementary Figure from Concurrent Inhibition of IGF1R and ERK Increases Pancreatic Cancer Sensitivity to Autophagy Inhibitors

Supplementary Data and Materials

Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK–MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K–AKT–mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor–based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC.Significance:CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.

The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) mandates the development of improved therapies. As KRAS mutations are found in 95% of PDAC and are critical for tumor maintenance, one promising strategy involves exploiting KRAS-dependent metabolic perturbations. The macrometabolic process of autophagy is upregulated in KRAS-mutant PDAC, and PDAC growth is reliant on autophagy. However, inhibition of autophagy as monotherapy using the lysosomal inhibitor hydroxychloroquine (HCQ) has shown limited clinical efficacy. To identify strategies that can improve PDAC sensitivity to HCQ, we applied a CRISPR-Cas9 loss-of-function screen and found that a top sensitizer was the receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R). Additionally, reverse phase protein array pathway activation mapping profiled the signaling pathways altered by chloroquine (CQ) treatment. Activating phosphorylation of RTKs, including IGF1R, was a common compensatory increase in response to CQ. Inhibition of IGF1R increased autophagic flux and sensitivity to CQ-mediated growth suppression both in vitro and in vivo. Cotargeting both IGF1R and pathways that antagonize autophagy, such as ERK–MAPK axis, was strongly synergistic. IGF1R and ERK inhibition converged on suppression of glycolysis, leading to enhanced dependence on autophagy. Accordingly, concurrent inhibition of IGF1R, ERK, and autophagy induced cytotoxicity in PDAC cell lines and decreased viability in human PDAC organoids. In conclusion, targeting IGF1R together with ERK enhances the effectiveness of autophagy inhibitors in PDAC.Significance:Compensatory upregulation of IGF1R and ERK–MAPK signaling limits the efficacy of autophagy inhibitors chloroquine and hydroxychloroquine, and their concurrent inhibition synergistically increases autophagy dependence and chloroquine sensitivity in pancreatic ductal adenocarcinoma.

Supplementary Tables

Although oncogenic driver mutations in RAS occur in 20% of cancers, heterogeneity in the biologic outputs of different RAS mutants has hampered efforts to develop effective treatments for RAS-mutated cancers. In this issue of Science Signaling, Huynh et al. show that even among KRAS(Q61) mutants, the specific amino acid that is substituted substantially affects mutant KRAS biologic activity and oncogenicity.

Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS—Gly 12 , Gly 13 , and Gln 61 (commonly known as G12, G13, and Q61, respectively)—occur differentially among the three RAS isoforms. Q61 mutations in KRAS are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively). Probability analysis suggested that mutational susceptibility to different DNA base changes cannot account for this distribution. Therefore, we investigated whether these frequencies might be explained by differences in the biochemical, structural, and biological properties of KRAS Q61 mutants. Expression of KRAS Q61 mutants in NIH 3T3 fibroblasts and RIE-1 epithelial cells caused various alterations in morphology, growth transformation, effector signaling, and metabolism. The relatively rare KRAS Q61E mutant stimulated actin stress fiber formation, a phenotype distinct from that of KRAS Q61H/R/L/P , which disrupted actin cytoskeletal organization. The crystal structure of KRAS Q61E was unexpectedly similar to that of wild-type KRAS, a potential basis for its weak oncogenicity. KRAS Q61H/L/R -mutant pancreatic ductal adenocarcinoma (PDAC) cell lines exhibited KRAS-dependent growth and, as observed with KRAS G12 -mutant PDAC, were susceptible to concurrent inhibition of ERK-MAPK signaling and of autophagy. Our results uncover phenotypic heterogeneity among KRAS Q61 mutants and support the potential utility of therapeutic strategies that target KRAS Q61 mutant–specific signaling and cellular output.

KRAS mutations occur in 95% of pancreatic ductal adenocarcinomas (PDAC) and are a well-validated driver of PDAC growth. Therefore, anti-KRAS therapies are expected to make a significant impact on the treatment of this deadly cancer, where there are currently no effective targeted therapies. Supporting this premise, early clinical trial results with KRASG12C inhibitors have shown promising disease control rates (84-100%) in KRASG12C-mutant PDAC. Despite these observations, two key issues limit the impact of KRASG12C inhibitors in PDAC. First, KRASG12C(OFF) mutations comprise less than 2% of KRAS mutations in PDAC. Second, patients initially responsive to KRASG12C inhibitors invariably relapse due to treatment-induced resistance. To begin qualifying KRAS inhibitors that target KRAS mutations more frequently found in PDAC, we characterized a RAS inhibitor that targets the multiple KRAS mutations as well as wild-type RAS proteins. We evaluated the impact of this inhibitor on RAS signaling and anti-proliferative activity in KRAS-mutant pancreatic cancer human cell lines and in a panel of RASless MEFs (ras-null mouse embryo fibroblasts) stably expressing exogenous RAS mutant proteins that are commonly found in PDAC. To identify genetic mechanisms of resistance to KRAS inhibition in pancreatic cancer, we applied CRISPR-Cas9 loss-of-function screens to KRASG12C-, KRASG12D-, KRASG12R-, and KRASQ61H-mutant PDAC cells treated with KRAS inhibitors to identify genes that modulate KRAS inhibitor anti-proliferative activity. We identified expected and novel mechanisms of resistance, including those that have been observed in patients treated with KRASG12C inhibitors. Citation Format: Andrew Michael Waters, Wen-Hsuan Chang, Clint Stalnecker, Cole Edwards, Runying Yang, Craig M. Goodwin, Adrienne D. Cox, Channing J. Der. Identification of resistance mechanisms to direct KRAS inhibition in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1075.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRASG12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (∼1%) in lung and colorectal cancers, yet relatively common (∼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRASG12R is functionally distinct from the more common KRASG12D- or KRASG12V-mutant proteins (KRASG12D/V). We found that KRASG12D/V but not KRASG12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRASG12D/V- but not KRASG12R-mutant PDAC. Surprisingly, we found that KRASG12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRASG12R-mutant PDAC. Finally, we determined that KRASG12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRASG12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy.Significance:We determined that KRASG12R is impaired in activating a key effector, p110α PI3K. As such, KRASG12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.See related commentary by Falcomatà et al., p. 23.This article is highlighted in the In This Issue feature, p. 1

The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) mandates the development of improved therapies. As KRAS mutations are found in 95% of PDAC and are critical for tumor maintenance, one promising strategy involves exploiting KRAS-dependent metabolic perturbations. The macrometabolic process of autophagy is upregulated in KRAS-mutant PDAC, and PDAC growth is reliant on autophagy. However, inhibition of autophagy as monotherapy using the lysosomal inhibitor hydroxychloroquine (HCQ) has shown limited clinical efficacy. To identify strategies that can improve PDAC sensitivity to HCQ, we applied a CRISPR-Cas9 loss-of-function screen and found that a top sensitizer was the receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R). Additionally, reverse phase protein array pathway activation mapping profiled the signaling pathways altered by chloroquine (CQ) treatment. Activating phosphorylation of RTKs, including IGF1R, was a common compensatory increase in response to CQ. Inhibition of IGF1R increased autophagic flux and sensitivity to CQ-mediated growth suppression both in vitro and in vivo. Cotargeting both IGF1R and pathways that antagonize autophagy, such as ERK–MAPK axis, was strongly synergistic. IGF1R and ERK inhibition converged on suppression of glycolysis, leading to enhanced dependence on autophagy. Accordingly, concurrent inhibition of IGF1R, ERK, and autophagy induced cytotoxicity in PDAC cell lines and decreased viability in human PDAC organoids. In conclusion, targeting IGF1R together with ERK enhances the effectiveness of autophagy inhibitors in PDAC.Significance:Compensatory upregulation of IGF1R and ERK–MAPK signaling limits the efficacy of autophagy inhibitors chloroquine and hydroxychloroquine, and their concurrent inhibition synergistically increases autophagy dependence and chloroquine sensitivity in pancreatic ductal adenocarcinoma.

Mutational activation of the KRAS oncogene is found in ~95% of pancreatic ductal adenocarcinoma (PDAC), the major form of pancreatic cancer. With substantial experimental evidence that continued aberrant KRAS function is essential for the maintenance of PDAC tumorigenic growth, the National Cancer Institute has identified the development of effective anti-KRAS therapies as one of four major initiatives for pancreatic cancer research. The recent clinical success in the development of an anti-KRAS therapy targeting one specific KRAS mutant (G12C) supports the significant potential impact of anti-KRAS therapies. However, KRAS

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X-mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X-dependency in RMS cells, however there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by co-suppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade, and particularly co-targeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X-mutant RMS tumor growth.

Human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide with an unmet need for more effective, less toxic treatments. Currently, both the disease and the treatment of HNSCC cause significant mortality and morbidity. Targeted therapies hold new promise for patients with HPV-negative status whose tumors harbor oncogenic HRAS mutations. Recent promising clinical results have renewed interest in the development of farnesyltransferase inhibitors (FTIs) as a therapeutic strategy for HRAS-mutant cancers. With the advent of clinical evaluation of the FTI tipifarnib for the treatment of HRAS-mutant HNSCC, we investigated the activity of tipifarnib and inhibitors of HRAS effector signaling in HRAS-mutant HNSCC cell lines. First, we validated that HRAS is a cancer driver in HRAS-mutant HNSCC lines. Second, we showed that treatment with the FTI tipifarnib largely phenocopied HRAS silencing, supporting HRAS as a key target of FTI antitumor activity. Third, we performed reverse-phase protein array analyses to profile FTI treatment-induced changes in global signaling, and conducted CRISPR/Cas9 genetic loss-of-function screens to identify previously unreported genes and pathways that modulate sensitivity to tipifarnib. Fourth, we determined that concurrent inhibition of HRAS effector signaling (ERK, PI3K, mTORC1) increased sensitivity to tipifarnib treatment, in part by overcoming tipifarnib-induced compensatory signaling. We also determined that ERK inhibition could block tipifarnib-induced epithelial-to-mesenchymal transition, providing a potential basis for the effectiveness of this combination. Our results support future investigations of these and other combination treatments for HRAS mutant HNSCC.

SUMMARY To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-β-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the βIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation., Graphical Abstract, In brief Using integrated analysis of genome-scale screens, Waters et al. identify BCAR1 and TUBB3, encoding p130Cas and βIII-tubulin, as two pancreatic cancer vulnerabilities that, when inhibited, sensitize KRAS mutant PDAC cells to ERK inhibition. Mechanistically, inhibition of p130Cas, βIII-tubulin, and ERK converges on and drives MYC loss to inhibit pancreatic cancer growth.

SUMMARY We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC., Graphical Abstract, In Brief Ozkan-Dagliyan et al. apply a chemical screen and identify concurrent inhibition of RAF and ERK as the most potent combination that causes low-dose apoptotic death of KRAS mutant pancreatic cancer. This vertical inhibition combination is resistant to loss of negative feedback mechanisms that reactivate ERK and potently suppresses diverse ERK-dependent processes.

To identify therapeutic targets for KRAS-mutant pancreatic cancer, we conducted a druggable genome siRNA screen and determined that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identified BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determined that SRC inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-beta-catenin dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the betaIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function, decreased levels of MYC protein and potently sensitized pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperated to reduce MYC protein and to synergistically suppress the growth of KRAS-mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

Rhabdomyosarcoma (RMS) is the most commonly diagnosed soft tissue sarcoma in children. While the 5-year event-free survival for localized rhabdomyosarcoma is over 70%, it is less than 30% for patients presenting with metastatic disease. Given that the current treatment modalities for RMS patients remain cytotoxic therapies with profound life-long lasting complications, identification of less toxic and more effective therapies is highly desired. Whole-genome and RNA sequencing of human rhabdomyosarcoma have identified frequent RAS genes oncogenic mutations in fusion-negative rhabdomyosarcoma (FN-RMS), which comprises 65% of RMS. This high incidence of RAS mutations present opportunity for design of targeted therapeutic strategies for FN-RMS. By siRNA-mediated knock down of individual RAS isoforms we showed that H/NRAS Q61X-mutant FN-RMS cell lines exhibited oncogenic RAS dependency and that knockdown of the Q61X-mutant H/NRAS inhibited ERK MAPK, but not PI3K-AKT pathway activity. Knockdown of the Q61X-mutant H/NRAS phenocopied ERK inhibitor treatment by inducing myogenic differentiation. Further, as compared to RAS-wild type RMS cells, H/NRAS-Q61X FN-RMS cell lines were preferentially more sensitive to MEK or ERK, but not PI3K or AKT inhibitors. RNA sequencing analysis revealed significant overlap of genes signatures associated with NRAS-Q61H knockdown and ERK inhibitor treatment in the FN-RMS cell line RD. These results indicated a central role of the ERK MAPK pathway in mediating H/NRAS Q61X-dependency in FN-RMS. However, in vivo evaluation of clinically relevant exposers with the MEK inhibitor trametinib demonstrated poor response, which correlated with inefficient ERK inhibition in pharmacodynamic assays. A trametinib-sensitizing CRISPR screen pointed to vertical inhibition of the RAF-MEK-ERK cascade by co-suppression of MEK and either CRAF or ERK. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically blocked ERK activity and induced cell cycle arrest, myogenic differentiation and cell death. In vivo evaluation of low dose pan-RAFi + ERKi or MEKi + ERKi potently suppressed H/NRAS Q61X RMS tumor growth, with pan-RAFi + ERKi being more effective and better tolerated, suggesting wider therapeutic window. These results support the clinical translation of pan-RAFi + MEKi/ERKi for H/NRAS Q61X-mutant FN-RMS. Citation Format: Natalia Garcia, Vanessa Del-Pozo, Marielle Yohe, Craig Goodwin, Terry Shackleford, Long Wang, Kunal Baxi, Yidong Chen, Myron Ignatius, Kris Wood, Peter Houghton, Angelina Vaseva. Vertical inhibition of the RAF MEK ERK cascade induces myogenic differentiation, apoptosis and tumor regression in H/NRAS Q61X mutant rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB251.

KRAS mutations are found in 95% of pancreatic ductal adenocarcinoma (PDAC), where they are key cancer drivers. Effective anti-KRAS therapeutics are thus anticipated to make a significant improvement in PDAC treatment. Despite the promise of inhibiting KRAS effector signaling, such inhibitors have not demonstrated significant activity as monotherapy, and there are concerns regarding potential normal tissue toxicity. Inhibitor combinations may overcome limitations of both efficacy and toxicity. We performed a 525-drug chemical biology screen to identify combinations that enhanced the cytotoxic activity of inhibitors of the RAF-MEK-ERK and the PI3K-AKT-mTOR effector pathways. Many such combinations were identified for RAF effector pathway inhibitors, while few were identified for PI3K pathway inhibitors. Generally, the same classes of inhibitors were identified for inhibitors of the RAF, MEK, and ERK nodes of the MAP kinase (MAPK) cascade. We found that inhibitors of the PI3K-AKT-mTOR pathway, microtubules, HDAC, and HSP90 each synergistically enhanced the cytotoxicity of RAF, MEK, and ERK inhibitors, in both conventional and PDX-derived PDAC cell lines. Mechanistically, we found that a shared basis for this synergy was enhanced loss of MYC protein, a key ERK substrate. Our studies identify promising combinations of KRAS effector inhibitors for PDAC treatment. Citation Format: Adrienne D. Cox, Craig M. Goodwin, Kirsten L. Bryant, Irem Dagliyan, Samuel D. George, Kelly E. Lucas, Prson Gautam, Krister Wennerberg, Channing J. Der. Inhibitor combinations targeting KRAS effector signaling in KRAS-mutant pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY20-02.

Significance Despite decades of research, no approved drugs have been discovered for KRAS. Recently, a pocket occurring on the surface of the active and inactive form of KRAS was found, but, due to its comparatively shallow, polar nature, this pocket has been assumed to be “undruggable.” Starting from very weakly binding fragments and using structure-based drug design, we discovered BI-2852 (1), a nanomolar inhibitor to this pocket which is mechanistically distinct to covalent KRASG12C inhibitors; 1 modulates pERK and pAKT and has an antiproliferative effect in KRAS mutant cells. This work demonstrates the druggability of this so-called switch I/II pocket and provides the scientific community with a chemical probe that directly inhibits the active and inactive forms of KRAS., The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be “undruggable,” between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.

SUMMARY We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC., Graphical Abstract, In brief Klomp et al. show that CHK1 is essential for KRAS mutant pancreatic cancer cell growth. CHK1 inhibition causes apoptotic growth suppression, MYC loss, and compensatory ERK and autophagy activation. Concurrent CHK1, ERK, and/or autophagy inhibition enhances apoptotic growth suppression. Additionally, genetic depletion of ERK-regulated DNA damage repair protein RIF1 phenocopies ERK inhibition.

Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl-1 inhibitors. This article is protected by copyright. All rights reserved.

Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins responsible for the regulation of programmed cell death (PCD). Amplification of Mcl-1 is a common genetic aberration in human cancer whose overexpression contributes to the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through interactions with pro-apoptotic BH3 containing proteins that achieve high affinity for the target by utilizing four hydrophobic pockets in its binding groove. Here we describe the discovery of Mcl-1 inhibitors using fragment based methods and structure-based design. These novel inhibitors exhibit low nanomolar binding affinities to Mcl-1 and greater than 500-fold selectivity over Bcl-xL. X-ray structures of lead Mcl-1 inhibitors when complexed to Mcl-1 provided detailed information on how these small-molecules bind to the target, and were used extensively to guide compound optimization.

RAS pathway mutations are found in nearly 75% of high-risk embryonal rhabdomyosarcoma (ERMS). While RAS oncoproteins are well-established therapeutic targets for many adult human cancers, still very little is known about the role of RAS mutations in the development and maintenance of ERMS. By sequencing, we identified cell lines and PDX tumors harboring activating mutations in H- or NRAS. Further, we showed that mutant H- or NRAS was critical for the growth of all RAS-mutant ERMS cell lines and that RAF/MEK/ERK signaling pathway, but not PI3K/AKT, was mediator of RAS dependency in these cells. However, in vivo treatment of RAS-mutant ERMS xenografts with the MEK inhibitor trametinib showed modest response as compared to BRAF-mutant astrocytoma xenografts. We reasoned that similarly to other RAS-driven cancers, ERMS cells and tumors are able to acquire resistance to inhibitors of the RAF/MEK/ERK pathway. We performed drug-sensitizing pooled CRISPR library screen and identified that inhibition of ERK2 potentiated trametinib treatment. We show that combining trametinib with ERK1/2 inhibitor leads to potent synergistic ERK inhibition and ERMS tumor growth suppression. Citation Format: Angelina V. Vaseva, Abhik Bandyopadhyay, Vanessa Del Pozo, Craig M. Goodwin, Prson Gautam, Krister Wennerberg, Kris C. Wood, Yidong Chen, Channing J. Der, Peter J. Houghton. Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A54.

Head and neck squamous cell carcinoma (HNSCC) affects more than 50,000 people annually. The five-year survival rate has not improved significantly in the last decades. In addition, many treatment modalities are associated with significant morbidity that negatively impacts survivors’ quality of life. Mutations in the HRAS oncogene are presented in 5-10% cases of HNSCC. Our group has established its critical importance for the growth and survival of HNSCC. HRAS protein undergoes crucial post-translational modifications prompted by the obligate addition of a farnesyl isoprenoid group, which is required for proper localization and insertion into the cell membrane, where it can engage effector molecules. FTIs block farnesylation and thus block HRAS activity. The FTI tipifarnib is under clinical evaluation for the treatment of HRAS mutant HNSCC, and preliminary findings showed some efficacy in this patient population. We sought strategies to enhance the efficacy of tipifarnib for the treatment of HRAS mutant HNSCC. We applied a novel CRISPR/Cas9 genetic screen to identify druggable targets that could be inhibited in combination with FTIs to improve treatment outcomes. We identified genes involved in regulating the ERK-MAPK and PI3K-AKT effector signaling pathways, autophagy regulation, chemokine signaling, and chromatin structure, that could potentially be targeted to increase tumor sensitivity to tipifarnib. Consistent with our genetic screening result of enhanced vulnerability to autophagy inhibition in tipifarnib-treated cells, we observed an increase in autophagic flux upon short-term treatment with tipifarnib alone, suggesting an attempt to compensate for nutrient stress. To overcome this effect, we tested the consequences of inhibiting autophagy pharmacologically in combination with tipifarnib. A panel of HRAS mutant HNSCC cell lines showed a synergistic increase in sensitivity to tipifarnib in combination with SBI-0206965 and MRT68921, two distinct preclinical inhibitors of the autophagy-promoting kinase ULK1. Clinical candidate ULK inhibitors are on the horizon. We suggest that the combination of tipifarnib with ULK inhibitors could be useful to sensitize HRAS mutant HNSCC to tipifarnib. Additional validation and mechanistic experiments are ongoing. Citation Format: Sehrish-Javaid, Craig M. Goodwin, Kirsten L. Bryant, Samuel D. George, Victoria V. Nguyen, Kathryn N. Lambert, Andrew M. Waters, Channing J. Der, Adrienne D. Cox. CRISPR/Cas9 genetic screen identifies novel therapeutic strategies for treating HRAS mutant HNSCC with farnesyltransferase inhibitors (FTIs) [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B15.

With KRAS mutations found in 95% of pancreatic ductal adenocarcinoma (PDAC), an effective anti-KRAS therapeutic strategy is anticipated to make a significant impact on the treatment of PDAC. Among the major directions currently being pursued, inhibitors of KRAS effector signaling are believed to be the most promising. However, as monotherapy, effector inhibitors have not demonstrated significant activity and concerns with normal tissue toxicity remain. Combination inhibitor strategies are considered the best approach to overcome these limitations. To identify effector inhibitor-based combinations, we applied a 525-oncology drug screen to identify combinations that enhanced the cytotoxic activity of inhibitors of the RAF-MEK-ERK and the PI3K-AKT-mTOR effector pathways. While many cytotoxic combinations were identified with RAF effector pathway inhibitors, few were identified with PI3K pathway inhibitors. The same classes of inhibitors were identified with RAF, MEK and ERK inhibitors. In addition to inhibitors of the PI3K-AKT-mTOR pathway, microtubule, HDAC and HSP90 inhibitors synergistically enhanced RAF and ERK inhibitor antitumor activity. Addressing a basis for synergy, we found that the different combinations showed enhanced loss of MYC protein, a key ERK substrate. In summary, our studies identify promising effector inhibitor-based combinations for PDAC treatment. Citation Format: Irem Ozkan-Dagliyan, Craig M. Goodwin, Kirsten L. Bryant, Samuel D. George, Kelly Lucas, Prson Gautam, Krister Wennerberg, Adrienne D. Cox, Channing J. Der. Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B31.

Breast cancer is the second-most frequently diagnosed malignancy in US women. The triple-negative breast cancer (TNBC) subtype, which lacks expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, afflicts 15% of patients and is refractory to current targeted therapies. Like many cancers, TNBC cells often deregulate programmed cell death by upregulating anti-apoptotic proteins of the B-cell CLL/lymphoma 2 (Bcl-2) family. One family member, myeloid cell leukemia-1 (Mcl-1), is commonly amplified in TNBC and correlates with a poor clinical prognosis. Here we show the effect of silencing Mcl-1 and Bcl-2-like protein 1 isoform 1 (Bcl-xL) expression on viability in a panel of seventeen TNBC cell lines. Cell death was observed in a subset upon Mcl-1 knockdown. In contrast, Bcl-xL knockdown only modestly reduced viability, indicating that Mcl-1 is a more important survival factor. However, dual silencing of both Mcl-1 and Bcl-xL reduced viability in most cell lines tested. These proliferation results were recapitulated by BH3 profiling experiments. Treatment with a Bcl-xL and Bcl-2 peptide had only a moderate effect on any of the TNBC cell lines, however, co-dosing an Mcl-1-selective peptide with a peptide that inhibits Bcl-xL and Bcl-2 was effective in each line tested. Similarly, the selective Bcl-xL inhibitor WEHI-539 was only weakly cytotoxic across the panel, but sensitization by Mcl-1 knockdown markedly improved its EC50. ABT-199, which selectively inhibits Bcl-2, did not synergize with Mcl-1 knockdown, indicating the relatively low importance of Bcl-2 in these lines. Mcl-1 sensitivity is not predicted by mRNA or protein levels of a single Bcl-2 family member, except for only a weak correlation for Bak and Bax protein expression. However, a more comprehensive index composed of Mcl-1, Bcl-xL, Bim, Bak and Noxa protein or mRNA expression correlates well with Mcl-1 sensitivity in TNBC and can also predict Mcl-1 dependency in non-small cell lung cancer cell lines.

Activating mutations in the HRAS oncogene are present in 5-11% of head and neck squamous cell carcinomas (HNSCC), but whether mutant HRAS drives HNSCC growth was not known. The clinical candidate FTI, tipifarnib, is currently under investigation in HRAS-mutant HNSCC, but whether patient responses to tipifarnib are due to blocking HRAS membrane association or due to action on another FTI target/s is also unclear. We found that the FTIs tipifarnib and lonafarnib inhibited the growth of HRAS-mutant HNSCC in a target-dependent manner, blocking HRAS farnesylation and membrane association. We also found that genetic depletion of mutant HRAS inhibited the anchorage-dependent and -independent growth of HRAS-mutant HNSCC cell lines in vitro and robustly induced apoptosis, indicating HNSCC dependence on mutant HRAS and a correlation between FTI-mediated growth inhibition and blockade of HRAS function. Tumor types driven by other RAS isoforms, such as KRAS-driven pancreatic cancer, are highly dependent on the ERK MAPK effector pathway and are sensitive to pharmacological inhibitors of ERK1/2 (ERKi: SCH772984, BVD-523). In contrast, we found that HRAS-driven HNSCC was resistant to growth inhibition by ERKi alone, despite strong target inhibition as indicated by decreases in pRSK and MYC protein levels. Consistent with this finding, genetic depletion of HRAS not only failed to reduce MAPK signaling but instead caused compensatory upregulation. Similarly, FTI treatment also induced upregulation of MAPK signaling, which is typically growth-promoting. We therefore speculated that the addition of ERKi would enhance responses to FTIs, and indeed found that ERKi synergistically enhanced FTI-mediated apoptosis and growth inhibition. Our results provide a mechanistic rationale for adding ERKi to FTI to improve clinical responses in HRAS-mutant HNSCC, and suggest that monitoring MAPK pathway activation may be a useful marker for emerging FTI resistance in this patient population. Citation Format: Sehrish Javaid, Victoria V. Nguyen, Andrew M. Waters, Craig M. Goodwin, Channing J. Der, Adrienne D. Cox. Farnesyltransferase inhibitor (FTI)-induced growth inhibition of HRAS-mutant head and neck cancers is enhanced by concurrently inhibiting compensatory upregulation of the ERK-MAPK kinase cascade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-276.

Pancreatic ductal adenocarcinoma (PDAC) patients have a dismal five-year survival rate of just eight percent in the advanced metastatic setting. Outcomes with standard chemotherapy regimens are less than ideal; therefore, the development of targeted therapies for the treatment of PDAC is a significant unmet clinical need. The two most frequent genetic events in PDAC (mutational activation of KRAS and loss of the tumor suppressor CDKN2A) converge on activation of the kinases CDK4 and CDK6, which promote G1 cell cycle progression. While clinically relevant agents directly targeting KRAS in PDAC remain elusive, pharmacologic restoration of CDKN2A function by inhibition of CDK4/6 may be an effective anti-KRAS therapeutic strategy. Supporting this hypothesis, we found that CDK4/6 inhibitors (e.g., palbociclib, abemaciclib, lerociclib) elicited single-agent activity in a subset of tested PDAC cell lines. However, using Reverse Phase Protein Array (RPPA), we observed widespread compensatory changes that overcome CDK4/6 inhibition, including increased anti-apoptotic protein expression, PI3K-mTOR and ERK MAPK signaling. Concurrent treatment with the potent and selective ERK1/2 inhibitor (ERKi) SCH772984 reversed resistance and increased sensitivity to palbociclib. This combination caused synergistic reduction in anchorage-dependent cell growth, and increased apoptosis, G1 arrest, and senescence. In an organoid model of pancreatic cancer, cell viability was synergistically reduced and caspase activation enhanced. We have also evaluated the combination of ERKi with palbociclib or the clinical-stage CDK4/6 inhibitor lerociclib (G1T38) in mouse models of PDAC, supporting our initiation of a phase Ib clinical trial of the ERK inhibitor ulixertinib/BVD-523 in combination with palbociclib in advanced metastatic pancreatic cancer (NCT03454035). Next, we sought to identify additional genes that regulate sensitivity to CDK4/6 inhibition. Using a CRISPR/Cas9 loss-of-function screen, we individually silenced expression of 2500 genes from the “druggable genome” in combination with palbociclib in six KRAS-mutant pancreatic and two KRAS-mutant colorectal cancer cell lines. We identified a wide array of genes that enhanced growth suppression in combination, centered around diverse signaling nodes including PI3K-AKT-mTOR signaling, cell cycle regulation and mitosis, SRC family kinase signaling, cell metabolism and biosynthesis, chromosome regulation and maintenance, and DNA damage & repair pathways, suggesting novel small molecule combinations to overcome de novo or acquired CDK4/6 inhibitor resistance in the clinic. We also identified genes whose loss imparts a survival advantage, suggesting possible resistance mechanisms to single-agent CDK4/6 inhibition. In total, these data suggest that CDK4/6 inhibitors alone, or in combination, may benefit PDAC patients clinically. Citation Format: Craig M. Goodwin, Sehrish Javaid, Andrew M. Waters, Bjoern Papke, Runying Yang, Mariaelena Pierobon, Daniel M. Freed, Patrick J. Roberts, Adrienne D. Cox, Kris C. Wood, Emanuel F. Petricoin, Autumn J. McRee, Channing J. Der. Combination therapies with CDK4/6 inhibitors to treat KRAS-mutant pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-287.

While mutational activation of KRAS occurs in nearly all pancreatic ductal adenocarcinomas (PDAC), clinically relevant agents directly targeting KRAS remain elusive. The second most frequent aberration in PDAC is loss of the tumor suppressor CDKN2A, which encodes INK4A, an endogenous inhibitor of the CDK4 and CDK6 cell cycle regulatory proteins that promote G1 cell cycle progression. KRAS and INK4A mutations cooperate to accelerate PDAC metastatic development, in part, by converging to block RB tumor suppressor activity. While no anti-KRAS targeted therapies have reached the clinic, pharmacologic restoration of CDKN2A function by inhibition of CDK4/6 may be an effective anti-KRAS therapeutic strategy. We found that CDK4/6 inhibitors (e.g., palbociclib) elicited single-agent activity in a subset of tested PDAC cell lines. Furthermore, concurrent treatment with the ERK inhibitor SCH772984 caused a potent synergistic reduction in anchorage-dependent and -independent cell growth, induced apoptosis, and increased senescence and G1 arrest. We next sought to identify novel genes that regulate sensitivity to palbociclib. Using a CRISPR/Cas9 loss-of-function screen, we individually silenced expression of 2500 genes from the “druggable genome” in combination with palbociclib. We identified genes that caused synergistic growth suppression in combination, suggesting small molecule combinations to overcome de novo or acquired CDK4/6 inhibitor resistance in the clinic. We also identified genes whose loss imparts a survival advantage, suggesting possible resistance mechanisms to single-agent CDK4/6 inhibition. In total, these data suggest that CDK4/6 inhibitors alone, or in combination, may benefit patients clinically. Citation Format: Craig Goodwin, Sehrish Javaid, Adrienne D. Cox, Kris Wood, Channing Der. Application of a CRISPR/Cas9 screen to identify novel therapeutic strategies with CDK4/6 inhibitors in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-276.

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family of proteins that is overexpressed and amplified in many cancers. Overexpression of Mcl-1 allows cancer cells to evade apoptosis and contributes to the resistance of cancer cells to be effectively treated with various chemotherapies. From an NMR-based screen of a large fragment library, several distinct chemical scaffolds that bind to Mcl-1 were discovered. Here, we describe the discovery of potent tricyclic 2-indole carboxylic acid inhibitors that exhibit single digit nanomolar binding affinity to Mcl-1 and greater than 1700-fold selectivity over Bcl-xL and greater than 100 fold selectivity over Bcl-2. X-ray structures of these compounds when complexed to Mcl-1 provide detailed information on how these small-molecules bind to the target, which was used to guide compound optimization.

Myeloid cell leukemia -1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which many cancers overexpress and depend on for survival. Mcl-1 inhibits apoptosis by binding to Bax and Bak, inhibiting the effectors of apoptosis from oligomerizing at the outer membrane of the mitochondria to promote cell death. Due to its role in cancer and resistance development to conventional chemotherapies, efforts to potently and specifically target MCL-1 have been ongoing. We have discovered novel small molecule inhibitors that selectively disrupt Mcl-1-Bim binding in the sub-nanomolar range. Anti-proliferative activities of our leading Mcl-1 inhibitors were assessed in a panel of hematological and solid cancer cell lines. We found that our compound promoted apoptosis in a subset of cell lines tested. As there was a variable response, we used BH3 profiling, which is a functional assay that rapidly measures dependence on any of the anti-apoptotic Bcl-2 family proteins for an individual sample to predict in vitro sensitivity to our Mcl-1 antagonists. Using the MS1 peptide, a known potent and selective Mcl-1 binder as a positive control in the BH3 profiling assay, we found a correlation between the BH3 profiling measurement of anti-apoptotic dependence to Mcl-1 and increased cytotoxicity caused by our compounds. These studies demonstrate that our compounds promote apoptosis through a selective inhibition of Mcl-1 mechanism of action and supports further investigation of our compounds in in vivo models of cancer. Citation Format: Leah Hogdal, John Sensintaffar, Allison Arnold, Craig Goodwin, Zhiguo Bian, Subrata Shaw, Chris Tarr, Taekyu Lee, Edward Olejniczak, Stephen Fesik. Predicting cellular response to small molecule Mcl-1 antagonists. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3726.

Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins that regulate apoptosis. Amplification of Mcl-1 is found in various cancers, which causes the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through high affinity interactions with pro-apoptotic BH3 containing proteins, Bak and Bax. Thus targeting Mcl-1 with small molecule inhibitors is a promising strategy but a very challenging task. Using fragment-based methods and structure-based design, we discovered a novel class of potent Mcl-1 inhibitors that exhibit selective anti-proliferative activity. New leads containing a tricyclic indole lactam scaffold exhibited dissociation constants of 1000-fold selectivity for Mcl-1 over Bcl-xL and Bcl-2. They also promoted apoptosis only in Mcl-1 sensitive cancer cell lines by activating caspases in a dose-dependent manner. These results provide a strong proof of concept for a selective inhibition of Mcl-1 function as an effective anti-cancer therapy. Finally, our leads also possess desirable pharmaceutical properties including in vivo oral bioavailability and represent an ideal starting point for developing clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers. Citation Format: Taekyu Lee, Zhiguo Bian, Johannes Belmar, Subrata Shaw, James C. Tarr, Bin Zhao, Nick Pelz, DeMarco Camper, Craig M. Goodwin, Allison L. Arnold, John L. Sensintaffar, Carrie F. Browning, Olivia W. Rossanese, Edward T. Olejniczak, Stephen W. Fesik. Discovery of orally bioavailable novel Mcl-1 inhibitors that exhibit selective anti-proliferative activity in Mcl-1 sensitive cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3551.

Mcl-1 (Myeloid cell leukemia 1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and is one of the major acquired resistance mechanisms for many anticancer therapies. Targeting Mcl-1 with small molecules is very challenging because Mcl-1 exerts its effects through protein-protein interactions. Here we describe our continuing efforts in the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. We previously described the discovery of small-molecule Mcl-1 inhibitors containing a 2-indole-carboxylic core that primarily bind to the P2 pocket. In order to enhance potency, we extend our molecules to occupy the entire BH3 binding interface. A number of P4 site hits were identified using a fragment-based screening while the P2 pocket of Mcl-1 was saturated with our previously reported lead. P4 site ligands were then linked to our initial lead guided by the ternary Mcl-1 structure containing both P2 and P4 units using an acylsulfonamide as a synthetic handle that retained the critical charged-charged interaction with R263. X-ray co-crystal structures of new inhibitors occupying both P2 and P4 sites bound Mcl-1 provided detailed information on how these small-molecules bind to the target and guided subsequent potency optimization. From the efforts, we discovered a novel series of 2-indole-acylsulfonamide containing Mcl-1 inhibitors that exhibit low nanomolar binding affinities to the target and greater than 500-fold selectivity over Bcl-xL. These compounds represent useful starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers. Citation Format: Subrata Shaw, James Chris Tarr, Nicholas Pelz, Zhiguo Bian, Johannes Belmar, Bin Zhao, Craig Goodwin, Allison Arnold, John Sensintaffar, Olivia Rossanese, Taekyu Lee, Edward Olejniczak, Stephen Fesik. Discovery of potent 2-Indole-acylsulfonamide Mcl-1 inhibitors using structure guided fragment-based methods. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4331.

Gas-solid chromatography was used to determine B(2s) (gas-solid virial coefficient) values for 12 alkanes (10 branched and 2 cyclic) interacting with a carbon powder (Carbopack B, Supelco). B(2s) values were determined by multiple size variant injections within the temperature range of 393 to 623 K with each alkane measured at 5 or 6 different temperatures. The temperature variations of the gas-solid virial coefficients were used to find the experimental adsorption energy or binding energy values (E( *)) for each alkane. A molecular mechanics based, rough-surface model was used to calculate the molecule-surface binding energy (E(cal)( *)) using augmented MM2 parameters. The surface model consisted of three parallel graphene layers with each layer containing 127 interconnected benzene rings and two separated nanostructures each containing 17 benzene rings arranged in a linear strip. As the parallel nanostructures are moved closer together, the surface roughness increases and molecule-surface interactions are enhanced. A comparison of the experimental and calculated binding energies showed excellent agreement with an average difference of 3.8%. Linear regressions of E( *) versus E(cal)( *) for the current data set and a combined current and prior alkane data set both gave excellent correlations. For the combined data set with 18 linear, branched and cyclic alkanes; a linear regression of E( *)=0.9848E(cal)( *) and r(2)=0.976 was obtained. The results indicate that alkane-surface binding energies may be calculated from MM2 parameters for some gas-solid systems.