Targeting RAS Research Articles

397 (PB385): High-Throughput 2D and 3D Cell Panel screening to facilitate RAS target drug Discovery and development

397 (PB385): High-Throughput 2D and 3D Cell Panel screening to facilitate RAS target drug Discovery and development

Targeted degradation of undruggable proteins using a novel heterobifunctional proteomimetic platform.

47 Background: Of the ~20,000 proteins in the human genome, only a subset can be modulated via traditional pharmacological approaches. “Undruggable” targets currently include proteins with pivotal roles in cancer pathogenesis. Our work aims to introduce a new paradigm for engaging these clinically relevant proteins using a modular platform we term the HYbrid DegRAding Copolymers (HYDRACs). HYDRACs multiplex targeting warheads with degradation inducers and have long circulation times plus high cell uptake. Preliminary studies on MYC and RAS, targets of great pharmaceutical interest, highlight the potential of this platform to revolutionize drug design. Methods: HYDRACs containing MYC or RAS targeting peptides randomly copolymerized with degrons were synthesized using ROMP. Fluorescent and biotin-tags were incorporated for use in uptake and pull-down assays. Viability following treatment with MYC-HYDRACs was performed in multiple cell lines with both cellular (MYC-independent lines) and polymer composition controls. Target engagement was assessed via immunoprecipitation and circular dichroism with protein degradation monitored by WB. Unbiased whole proteome analysis and RNAseq were done to confirm observed effects were selectively on-target. In vivo efficacy and biodistribution was assessed in tumor-bearing mice. Results: HYDRACs show high levels of cell uptake and antiproliferative effects at sub-micromolar concentrations in a formulation- and target-dependent manner. Cells treated with MYC-HYDRACs displayed reduced MYC protein levels, which were rescued by proteasome or neddylation inhibition. Swapping out the degron for various validated E3 ligase recruiters maintained MYC degradation, highlighting the “plug-and-play” nature of this approach. On-target activity was confirmed via RNAseq. Biophysical analysis showed strong HYDRAC-protein interactions, orthogonally supported by the presence of target protein following pull-down of biotin-terminated HYDRACs. Target protein degradation only occurred following treatment with an intact HYDRAC compound. Mice bearing MYC-CaP tumors showed delayed tumor growth following treatment with MYC-HYDRACs, with compound accumulation in the tumor for up to 72 h following a single injection. Generalizability was demonstrated against RAS, with RAS-HYDRACs capable of degrading KRAS across multiple different alleles, acting as a pan-KRAS degrader. Conclusions: We present a novel platform technology that addresses the challenges inherent to peptide delivery approaches. HYDRACs have the potential to dramatically alter the drug discovery landscape, allowing for the development of cancer-relevant target modulators for which there are no current viable therapies. We envision the HYDRAC platform as a generalizable approach to designing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for TPD.

Finding the sweet spot: Targeting RAS in tumors while sparing normal tissue

The development of mutant-selective KRAS inhibitors represents a major therapeutic advance; however, patients can develop resistance through feedback mechanisms and genetic alterations in the RAS pathway. Three publications in Nature and Cancer Discovery describe a promising RAS(ON) multi-selective inhibitor that simultaneously targets oncogenic RAS and multiple potential resistance mechanisms while sparing normal tissue.

Targeting KRAS Oncogene for Patients With Colorectal Cancer: A New Step Toward Precision Medicine.

KRAS mutations are common driver oncogenes associated with the development of several solid tumors. KRAS oncogene has been considered a highly challenging target for drug development because of structural features, including the lack of deep groove on its catalytic unit. However, by leveraging cysteine residues, covalent KRAS inhibitors irreversibly trap KRAS G12C mutants in their inactive GDP-bound state. These agents have resulted in significant clinical responses among patients with KRAS G12C-mutant solid tumors, including patients with colorectal cancer (CRC). Other allele-specific inhibitors of KRAS oncogene and panKRAS and panRAS inhibitors are also currently being investigated in clinical trials. This review article overviews recent clinical progress on KRAS G12C targeting for the management of patients with KRAS G12C-mutant CRC and provides an update on other RAS targeting approaches. We also discuss the unique biological features of RAS-mutant CRC, which require the combination of KRAS inhibitors and anti-epidermal growth factor receptor therapy, and elaborate on resistance mechanisms and novel therapeutic avenues that may define future treatment paradigms of patients with RAS-mutant CRC.

State-of-the-art and upcoming trends in RAS-directed therapies in gastrointestinal malignancies.

Overall, the review underscores the evolving landscape of KRAS-targeted therapy and the potential for these approaches to improve outcomes for patients with gastrointestinal malignancies. It highlights the importance of ongoing research and clinical trials in advancing precision medicine strategies for KRAS-driven cancers. This review provides a comprehensive overview of the RAS signaling pathway and its significance in gastrointestinal malignancies. The introduction of KRAS inhibitor represents a significant advancement in the treatment landscape for KRAS-mutant cancers. In this review, we discuss upcoming trends in KRAS-targeted therapy, including the development of mutant-specific direct KRAS inhibitors like MRTX1133 and pan-RAS inhibitors such as RMC-6236. It also explores indirect RAS inhibitors targeting upstream and downstream components of the RAS pathway. Additionally, the review examines other upcoming strategies like combination therapies, such as CDK4/6 and ERK MAPK inhibitors, as well as adoptive cell therapy and cancer vaccines targeting KRAS-mutant cancers. Targeting RAS has become an important strategy in treating gastrointestinal cancer. These findings in this review underscore the importance of a multidisciplinary approach, integrating advances in molecular profiling, targeted therapy, immunotherapy, and clinical research to optimize treatment strategies for patients with KRAS-mutant gastrointestinal malignancies.

Targeting KRAS in cancer.

RAS family variants-most of which involve KRAS-are the most commonly occurring hotspot mutations in human cancers and are associated with a poor prognosis. For almost four decades, KRAS has been considered undruggable, in part due to its structure, which lacks small-molecule binding sites. But recent developments in bioengineering, organic chemistry and related fields have provided the infrastructure to make direct KRAS targeting possible. The first successes occurred with allele-specific targeting of KRAS p.Gly12Cys (G12C) in non-small cell lung cancer, resulting in regulatory approval of two agents-sotorasib and adagrasib. Inhibitors targeting other variants beyond G12C have shown preliminary antitumor activity in highly refractory malignancies such as pancreatic cancer. Herein, we outline RAS pathobiology with a focus on KRAS, illustrate therapeutic approaches across a variety of malignancies, including emphasis on the 'on' and 'off' switch allele-specific and 'pan' RAS inhibitors, and review immunotherapeutic and other key combination RAS targeting strategies. We summarize mechanistic understanding of de novo and acquired resistance, review combination approaches, emerging technologies and drug development paradigms and outline a blueprint for the future of KRAS therapeutics with anticipated profound clinical impact.

Abstract 5835: Genome-Wide CRISPR gene knockout screens combined with selpercatinib identifies potent combination therapies for RET driven medullary thyroid carcinoma

Abstract Background: Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor driven primarily by activating mutations in the RET proto-oncogene. It accounts for approximately 13% of thyroid cancer-related deaths. Total thyroidectomy is the main treatment option for MTC patients; however, its efficacy is limited due to the high prevalence of metastatic disease at diagnosis. Vandetanib, cabozantinib, and selpercatinib are RET signaling inhibitors (RETi), that have been approved for treating advanced MTC. Despite their effectiveness, many patients relapse or have refractory disease due to eventual resistance to these RETis. Methods: In our study, we employed genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) gene knockout (KO) in MTC cell lines treated with selpercatinib or vehicle to investigate the genetic mechanisms of resistance or sensitivity to RETi. Our overarching goal was to identify potential synergistic or additive drug combinations that may prevent or treat resistant disease. Results: Our research identified key genes and pathways confirming the sensitivity or resistance of MTC cells to RETi. Notably, apoptotic genes along with RAS and mTOR signaling pathways were found to play a significant role in resistance development. Knocking out negative regulators of these pathways (such as NF1 and TSC2) conferred growth advantages to cells treated with RETi. Alternatively, knocking out BCL2, an antiapoptotic protein, led to increased lethality. These findings suggested that targeting RAS, mTOR, or antiapoptotic pathways in conjunction with RETi may be synergistic or have additive activity. To validate the CRISPR screening, we selectively knocked down NF1 in MTC cells and found it led to a nearly two-fold increase in IC50 for selpercatinib. Furthermore, in-vitro and in-vivo testing confirmed significant synergistic activity when using the combination of trametinib, an MEK inhibitor (MEKi; downstream of the RAS pathway), with selpercatinib. Conclusions and Future Directions: We have identified a potent synergistic combination of MEKi with RETi as a novel synergistic drug combination to treat MTC, which may also prevent resistance to RETi. These results will be translated to patients through clinical trials conduced at the NCI. Citation Format: Abdelrahman Rahmy, Arwa Fallatah, David Milewski, Meijie Tian, Young Song, Yong Kim, Hsien-Chao Chou, Xinyu Wen, Chaoyu Wang, Jun Wei, Robert Hawley, John Glod, Javed Khan. Genome-Wide CRISPR gene knockout screens combined with selpercatinib identifies potent combination therapies for RET driven medullary thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5835.

Abstract C087: Window-of-opportunity trial of metastatic pancreatic cancer reveals mechanisms of response to targeting RAS signaling

Abstract KRAS is mutated in ~85% of pancreatic ductal adenocarcinoma (PDAC) patients' tumors, suggesting that it is a key node of PDAC targeted therapy. RAF/MEK/ERK signaling is downstream of KRAS signaling. Preclinical studies targeting MEK showed promises but clinical trials failed to show the benefit compared to Standard-of-Care treatment chemotherapy. To elucidate the mechanism of action and resistance to several targeted therapies including MEK inhibition in human PDAC, we conducted a Window-Of-Opportunity trial for Metastatic pancreatic cancer (WOOM trial). In this trial, metastatic PDAC patients received 10 days of Cobimetinib, a FDA approved MEK inhibitor, therapy and specimens of pre- and post- treatment were obtained by biopsy to evaluate the response to targeted treatment using deep multi-omic analytics including DNA-seq, RNA-seq, multiplex IHC, cyclic IF and Digital Spatial Profiling (DSP). The clinical grade KI67 staining positivity was used as a readout of tumor response. We found three of 15 patients’ tumors had significantly decreased KI67 staining after MEKi treatment. Genomic sequence showed that two of three KI67-decreased patient’s tumors had KrasG12R mutations, which is significantly higher penetration compared to none of twelve KI67-non-responsive patient’s tumors. RNA-seq analysis showed that these KI67-decreased tumors are more classical-like compared to Ki67-non-responsive tumors. DSP analysis confirmed the downregulation of ERK phosphorylation after treatment in all examined cases. Moreover, MYC protein expression change is significantly correlated with KI67 protein expression change, which suggests MYC downregulation is the mechanism of action of MEK-inhibitor. Interestingly, MEK phosphorylation was upregulated after treatment in all examined cases. This suggests paradoxical RAF activation as a potential resistance mechanism. Furthermore, we found that Cobimetinib treatment changed the tumor microenvironment. Here, we summarize the multi-omics data and discuss the mechanism of response and resistance to target RAS signaling and related microenvironmental changes. Citation Format: Motoyuki Tsuda, Colin J. Daniel, Carl Pelz, Sam Sivagnanam, Dove Keith, Christopher L. Corless, Koei Chin, Lisa M. Coussens, Jonathan R. Brody, Charles D. Lopez, Gordon B. Mills, Rosalie C. Sears. Window-of-opportunity trial of metastatic pancreatic cancer reveals mechanisms of response to targeting RAS signaling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C087.

Abstract C074: Clinico-genomic characterization of N=2,460 pancreatic adenocarcinoma identifies KRASMUT dosage as prognostic of overall survival across disease stages

Abstract Background: The majority (~80%) of patients (pts) with pancreatic adenocarcinomas (PDAC) present with advanced disease. Most sequencing efforts to date have focused on early stage disease. Herein, we leverage a large clinical sequencing cohort of N= 2460 pts with PDAC to characterize the clinico-genomic landscape across different disease stages. Methods: Tumors from 2460 pts were sequenced using MSK-IMPACT clinical sequencing assay and mutations called via CLIA-approved pipelines. Detailed clinical descriptors including lines of treatment and response were abstracted for N=1451 (59%) pts. Intratumor heterogeneity estimates including allele-specific copy number were inferred using FACETS. Results: Of N=2460, at diagnosis 34% of pts had resectable tumors, and 24% and 42% had locally advanced and metastatic disease, respectively. 94% of pts had KRASMUT tumors. Expectedly, MAPK pathway alterations were significantly enriched in KRASWT tumors (60% vs. 7% in KRASMUT, p < 0.001) with gain-of-function kinase oncogene fusions exclusively observed in these tumors (p<0.001). In addition, GNAS, PTEN, FOXP1, SMARCB1 and CREBBP were enriched in the KRASWT tumors (q < 0.05). Collectively, pts with KRASWT/MAPK MUT and KRASWT/MAPK WT tumors comprise 4% and 2% of all pts PDAC, respectively. Germline pathogenic variants in high/moderate penetrance genes were more common in pts with KRASWT tumors compared to pts with KRASMUT tumors (20% vs. 10%, p < 0.001). Pathogenic germline alterations in BRCA1/2 and PALB2 were observed in 6% (N=144) of cohort. Biallelic inactivation of BRCA2 specifically was associated with prolonged response of metastatic pts to PARP-inhibitor maintenance therapy with 10.3 months on treatment prior to progression compared to 3.4 months for those with biallelic inactivation of other HRD genes and 2.2 months for those with non-biallelic inactivation. Among KRASMUT tumors assessed for KRAS copy-number allelic imbalance (AI) (N=1097), 41% (N=452) had AI at the KRAS locus with mutant-allele selected for in 94% of tumors with imbalance. Among KRASMUT tumors with normal genome ploidy, overall, 20% had a mutant allele gain ranging from 10% in resectable to 29% of metastatic tumors. We observe significantly lower OS among pts with KRASMUT dosage gain compared to heterozygous KRASMUT across pts with resectable (HR: 0.66, 95% CI: 0.18-1.15, p<0.01) and metastatic (HR: 0.39, 95% CI: 0.16-0.63, p=0.001) diseases with statistical significance not reached among the locally advanced disease (HR: 0.4, 95% CI: -0.01-0.81, p=0.056). Notably, the OS of pts with loss of the wild-type allele and no mutant copy gain was indistinguishable from that of heterozygous KRASMUT tumors (HR: -0.11, 95% CI: -0.37-0.15, p=0.4). Conclusion: In this large cohort of 2460 pts with PDAC, we observed that KRASMUT dosage gain is a significant negative predictor of OS. These findings highlight the importance of incorporating allelic-dosage into comprehensive molecular testing in PDAC given prognostic importance and the emerging era of direct RAS targeting. Citation Format: Anna M. Varghese, Maria A. Perry, Daniel Muldoon, Subhiksha Nandakumar, Amanda Erakky, Bastien Nguyen, Amanda Zucker, Hulya Sahin Ozkan, Olca Basturk, Debyani Chakravarty, David P. Kelsen, Wungki Park, Kenneth H. Yu, Alice C. Wei, Nikolaus Schultz, Michael F. Berger, Christine Iacobuzio-Donahue, Chaitanya Bandlamudi, Eileen M. O'Reilly. Clinico-genomic characterization of N=2,460 pancreatic adenocarcinoma identifies KRASMUT dosage as prognostic of overall survival across disease stages [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C074.

Progress in Targeting KRAS Directly.

RAS research has entered the world of translational and clinical science. Progress has been based on our appreciation of the role of RAS mutations in different types of cancer and the effects of these mutations on the biochemical, structural, and biophysical properties of the RAS proteins themselves, particularly KRAS, on which most attention has been focused. This knowledge base, while still growing, has enabled creative chemical approaches to targeting KRAS directly. Our understanding of RAS signaling pathways in normal and cancer cells plays an important role fordeveloping RAS inhibitors but also continues to reveal new approaches to targeting RAS through disruption of signaling complexes and downstream pathways.

Synergistic Anti-Tumor Effects in RAS Mut Multiple Myeloma By Targeting MAP4K2 and RAS Pathways

Mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) is an important key regulator of the stress-activated MAPK core signaling pathways. Our previous work demonstrated that MAP4K2 is highly expressed in multiple myeloma (MM) cells, and MM cells carrying a RAS mutation are more vulnerable to MAP4K2 inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins ( Li et al. Blood 2021). Targeting the aberrant RAS/MAPK signaling, MEK inhibitors have been shown to induce cytotoxicity in MM (De la Puente, P. et al. Blood Cancer J. 2016). Thus, we used a recently developed MEK inhibitor (MEKi) Trametinib to investigate the therapeutic potential of combined MEK and MAP4K2 signaling inhibition in RAS-driven MM. Tet-on sh-MAP4K2 lentivirus was introduced into RAS Mut MM cells to establish the inducible MAP4K2 knockdown cells upon doxycycline treatment. To examine the combined effects, Tet-on sh-MAP4K2 RAS Mut MM cells were treated with doxycycline to silence MAP4K2 expression, followed by treatment of MEKi Trametinib at different dosages. Cell proliferation, apoptosis, and cell cycle were analyzed five days later. Data showed that MAP4K2 silencing combined with MEK inhibition significantly decreased cell proliferation to 10% compared with MEKi alone 32% ( p<0.01) by MTS assays, and enhanced cell apoptosis (MEKi alone vs. with MAP4K2 silencing: 26% vs 91%, p<0.01). Cell cycle assay demonstrated that MEKi induced G1 arrest in RAS Mut MM cells (MEKi alone:76% vs vehicle control: 54%, p<0.01), which was also significantly higher in combination with MAP4K2 silencing (92%). Western blot assay confirmed that MAP4K2 silencing combined with MEKi significantly enhanced the downregulation of IKZF1 and c-MYC compared to the MEKi treatment alone. These data suggest that the MEKi and MAP4K2 inhibition combination has synergetic anti-MM effects. To further confirm the synergetic effects, we tested the combined effects of MEKi with the MAP4K2 kinase inhibitor TL4-12. H929 (N-Ras Mut) cells were treated by vehicle control, TL4-12 (2 uM) alone, MEKi (200 nM) alone, or TL4-12 + MEKi for 4 days, followed by an apoptosis assay. The combination of TL4-12 and MEKi strongly enhanced the cytotoxicity effects compared to the single-agent treatments. Consistently, the carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation assay confirmed that dual treatment significantly delayed H929 cell proliferation compared to the single-agent treatments (percentage of proliferating cells: TL4-12 alone: 86.2%; MEKi alone: 89.03%; dual treatment: 18.9%. p<0.01). Western blotting assay further confirmed that dual treatment of MEKi and MAP4K2i enhanced the decrease in MEK1/2 and ERK1/2 phosphorylation and downregulation of transcriptional factors IKZF1 and c-MYC. Taken together, our findings demonstrate that the combination of MAP4K2 inhibition with MEKi results in synergetic anti-MM effects in RAS mutation myeloma, therefore could be a potent novel therapeutic regimen for patients with relapsed/refractory multiple myeloma.

Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable.

Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Because clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC/RNA sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2,500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NOD/SCID gamma mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Because inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL/MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL/MAPK. We report on a novel mechanism of AXL/MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN.

Proteomic Mapping of the Interactome of KRAS Mutants Identifies New Features of RAS Signalling Networks and the Mechanism of Action of Sotorasib.

RAS proteins are key regulators of cell signalling and control different cell functions including cell proliferation, differentiation, and cell death. Point mutations in the genes of this family are common, particularly in KRAS. These mutations were thought to cause the constitutive activation of KRAS, but recent findings showed that some mutants can cycle between active and inactive states. This observation, together with the development of covalent KRASG12C inhibitors, has led to the arrival of KRAS inhibitors in the clinic. However, most patients develop resistance to these targeted therapies, and we lack effective treatments for other KRAS mutants. To accelerate the development of RAS targeting therapies, we need to fully characterise the molecular mechanisms governing KRAS signalling networks and determine what differentiates the signalling downstream of the KRAS mutants. Here we have used affinity purification mass-spectrometry proteomics to characterise the interactome of KRAS wild-type and three KRAS mutants. Bioinformatic analysis associated with experimental validation allows us to map the signalling network mediated by the different KRAS proteins. Using this approach, we characterised how the interactome of KRAS wild-type and mutants is regulated by the clinically approved KRASG12C inhibitor Sotorasib. In addition, we identified novel crosstalks between KRAS and its effector pathways including the AKT and JAK-STAT signalling modules.

RAS and Other Molecular Targets in Pancreatic Cancer: The Next Wave Is Coming.

Since the discovery of oncogenes in the 1970s, cancer doctors and researchers alike have understood the promise of discovering drugs to block the dominantly acting function of mutated signaling proteins in cancer. This promise was delivered, first slowly, with early signals inhibiting HER2 and BCR-Abl in the 1990s and 2000s, and then quickly, with kinase inhibitors being approved hand over fist in non-small cell lung cancer, melanoma, and many other malignancies. The RAS proteins, however, remained recalcitrant to chemical inhibition for decades, despite being, by far, the most frequently mutated oncogenes in cancers of all types. Nowhere was this deficit more palpable than in pancreatic ductal adenocarcinoma (PDA), where > 90% of cases are driven by single nucleotide substitutions at a single codon of the KRAS gene. The ice began to crack in 2012 when Ostrem and colleagues (Nature 503(7477): 548-551, 2013) synthesized the first KRAS G12C inhibitors, which covalently bind to GDP-bound G12C-mutated KRAS and lock the oncoprotein in its inactive state. In the last decade, the scientific community has established a new foundation on this and other druggable pockets in mutant KRAS. Here we provide an up-to-date overview of drugs targeting KRAS and other molecular targets in pancreatic cancer.

Genomic and transcriptomic profiling of inflammatory breast cancers and non-inflammatory breast cancers in Asian women.

e12546 Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Despite its rare incidence, it accounts for 10% of breast cancer-caused mortality. Previous studies based on NGS have focused on western populations and found no common genomic and transcriptomic difference that could distinguish IBC from non-IBC. Methods: We performed WES on 15 IBC samples and their paired normal blood to identify somatic genomic alterations, copy number variants and large structural variants. RNA-seq was performed on 16 IBC samples to examine the RNA expression, pathway enrichment and gene fusions. WES and RNA-seq data were further investigated to discover genes that were dysregulated in both genomics and transcriptomics. For comparison, we performed WES and RNA-seq on 5 non-IBC samples and acquired additional 33 non-IBC samples of Asian women from the Cancer Genome Atlas Program (TCGA). Results: Apart from results similar to the previous reports in western IBC, ZNF74 and DYNC2H1 were identified as Asian-IBC-specific somatic mutations in our cohort. Elevated HRD mutational signature and scarHRD score indicated increased HRD events in Asian IBC patients. Structural variation analysis showed that deletions were found more frequent in IBC. RNA-seq indicated increased immune activation in IBC samples, but the immune cell infiltration within the tumor microenvironment were similar between IBC and non-IBC. Multiple gene fusions were more frequently found in IBC, including CTC-786C10.1—RP11-680G10.1, TULP4—RP11-732M18.3, RN7SL4P—RN7SL173P. Combined analysis of WES and RNA-seq showed more frequent mutation and increased pathway enrichment of RAS pathway in IBC, which may explain the aggressive biological behavior of IBC, inhibitors such as sorafenib, tipifarnib and trametinib targeting RAS signaling and its downstream pathways were predicted to possess promising effects in IBC treatment. Conclusions: Our study is the first to provide a complete genomic and transcriptomic landscape of IBC based on combined analysis of WES and RNA-seq and the first to focus on Asian women. We discovered intriguing differences unique in Asian women that could potentially explain IBC etiology and presented RAS signaling pathway as a potential therapeutic target in IBC treatment.

Abstract A032: Targeting RAS beyond KRAS, a review

Abstract RAS proteins belong to the family of GTPases and plays instrumental role in cellular growth and proliferation and dysregulation of RAS signalling is one of the key drivers of tumorigenesis. The RAS protein has four isoforms namely KRAS4A, KRAS4B, NRAS and HRAS with amino acid codons 12, 13 and 61 as common hotspots for RAS mutations. Each of these hotspot mutations appears across several cancer types, however each of them have been noted to have specific distribution across different tissues. For example, mutations in codon 12 of KRAS are most common in pancreatic, lung and colorectal cancer, NRAS mutations affecting Q61 and G12 are most prevalent in melanoma and haematological malignancies, respectively, and HRAS mutations are more frequently altered in thyroid, bladder and head and neck cancers. Substantial efforts have been made to develop small molecule inhibitors targeting KRASG12C, with demonstrated success in clinic. Early success with KRASG12C has prompted researchers targeting other RAS isoforms, such as H-RAS and N-RAS to address RAS mutated cancer burden. Discovery of pan-RAS monobody that binds to all RAS isoforms have opened new opportunities. Development of small molecule inhibitors of RAS membrane association is gaining potential as a therapeutic strategy to curb RAS signalling; Lonafarnib and Tipifarnib, inhibitors of farnesyltransferase that lipidate Ras to facilitate membrane localization, have shown effectiveness in HRAS mutated cancers and are in active clinical evaluation. Although the lack of a hydrophobic binding pocket in NRAS poses challenges to drug targeting, specific strategies such as amphiphile-mediated depalmitoylation to target the post-translational modification of NRAS and reduce its activity are being investigated. The wide variety of approaches to target the RAS signalling pathway, beyond KRAS, have resulted in a broadening patent landscape. While finding ways to specifically target RAS mutants remains a daunting challenge, herculean efforts to make clinically useful RAS-targeted drugs are beginning to provide a bounty of weapons to fight against RAS-driven tumors. Citation Format: Neetu Singh, Darren R. Tyson, Partha Pratim Sarma, Sathish Katike, Prashant K. Bhavar, Uday Kumar Surampudi. Targeting RAS beyond KRAS, a review [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A032.

Abstract A006: Chromatin remodeling as a potential epigenetic mechanism of tolerance to KRAS loss

Abstract KRAS-targeted inhibition yielded promising, yet far from ideal, clinical responses, revealing that cancer cells easily bypass KRAS loss. Therefore, we aim to understand how mutant KRAS cancer cells tolerate so well the loss of a key oncogene to which they were addicted to. KRAS expression was silenced through siRNAs in colorectal cancer (CRC) cell lines that carry KRAS mutations. The proteome characterization was obtained by mass spectrometry and the expression of significantly altered proteins was validated by western blotting. Chromatin states were investigated using electron microscopy. Upon KRAS inhibition, there was a significant reduction in the cell number, accompanied by changes in cell cycle, thus supporting KRAS-dependency. Proteomics analysis revealed that KRAS inhibition-tolerant cells upregulated several proteins associated with the extracellular exosome or nuclear compartments. Molecular function and biological process gene ontology terms revealed an up-regulation of proteins mainly associated with binding activities (RNA, protein, nucleosome, and core promoter binding) as well as gene expression regulation, mRNA splicing and processing, and nucleosome assembly and repositioning. In addition, the proteomics data also revealed an upregulation of proteins associated with active chromatin states. Moreover, KRAS-silenced persister cells also presented alterations in some histone post-translational modifications and in chromatin packing, both suggesting that transcription is impacted. Overall, our results suggest an epigenetic mechanism underlying tolerance to KRAS inhibition that involves chromatin structural changes and transcription alterations, which we are currently pursuing. Citation Format: Flavia Martins, Ana L. Machado, Patricia D. Carvalho, Joana Carvalho, Rune Matthiesen, Vadim Backman, Sergia Velho. Chromatin remodeling as a potential epigenetic mechanism of tolerance to KRAS loss [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A006.

Abstract A022: Translation initiation factor 2B (eIF2B) stimulates mutant KRAS function in cancer

Abstract KRAS mutations appear with high frequency in colorectal, lung and pancreatic cancers, which are the three leading causes of new cancer deaths worldwide. Mutant KRAS is preferentially bound to GTP resulting in continuous cell proliferation. Mutant KRAS exposes cells to oncogenic forms of stress (i.e. genotoxic, metabolic, proteostatic stress), which disrupt proliferation and tissue homeostasis. To cope with stress, cells engage pro-adaptive mechanisms, which act in favor of mutant KRAS to transform cells. An important adaptation mechanism to stress acts at the level of mRNA translation and involves the functional interplay between the translation initiator factors eIF2 and eIF2B. Phosphorylated eIF2 mediates a translational and transcriptional reprogramming to promote adaptation under stress, a process that is antagonized by the guanine exchange function (GEF) of eIF2B. We demonstrate the physical interaction between mutant KRAS and eIF2B by mass spectrometry. Using genetic approaches, we show that eIF2B is required for the survival and proliferation of tumor cells with KRAS mutations via the stimulation of MAPK signaling. We also show that eIF2B contributes to increased resistance of tumor cells to pharmacological inhibition of mutant KRAS forms. Genetic inactivation of eIF2B promotes the formation of mutant KRAS-GDP complexes whereas its pharmacological stimulation facilitates mutant KRAS-GTP complex formation in tumor cells; this data supports a potential GEF function for eIF2B towards mutant KRAS. Cell imaging experiments provide strong evidence for the implication of eIF2B in the association of mutant KRAS with the plasma membrane of tumor cells. Our findings reveal a stimulatory role of eIF2B in mutant KRAS signaling and provide a previously unidentified link between mutant KRAS and mRNA translation with implications in the growth and treatment of cancers with KRAS mutations. Citation Format: Hyungdong Kim, Nour Ghaddar, Laleh Ebrahimi Ghahnavieh, Shuo Wang, Kwang-Jin Cho, Atsuo Sasaki, Antonis E. Koromilas. Translation initiation factor 2B (eIF2B) stimulates mutant KRAS function in cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A022.

Abstract B033: Wild-type RAS signaling is an essential therapeutic target in RAS-mutated cancers

Abstract Single-agent targeting of RAS-mutated cancers using RAF/MEK/ERK or PI3K/AKT pathway inhibitors is ineffective; blocking one pathway relieves negative feedback control of RTK signaling and hyperactivates parallel effector pathways. While combined MEK and PI3K inhibition is effective in preclinical models, toxicity of this combination prevents its clinical use. Alternative strategies for treating RAS-mutated cancers are essential. Therapeutics directly targeting mutated RAS proteins have the potential to spare normal cellular function thereby lessening overall toxicity. Unfortunately, direct RAS inhibition as a monotherapy does not fully block RAS effector signaling. RAS proteins show differential activation of RAF and PI3K pathways: KRAS potently activates RAF but poorly activates PI3K, whereas HRAS effectively activates PI3K but poorly activates RAF. Further, mutant RAS inhibition relieves negative feedback controls leading to rapid hyperactivation of RTK−WT RAS signaling. A more comprehensive understanding of the interplay between mutant RAS and RTK−WT RAS signaling is essential to developing rational therapeutic approaches to treat RAS-mutated cancers. We found that WT RAS enhanced mutant RAS-driven transformation by activating RAS effectors poorly engaged by mutated RAS. Further, inhibition of RAS effectors activated poorly by mutant RAS synergized with and limited resistance to mutant HRAS and KRAS inhibitors. The mutant HRAS inhibitor tipifarnib blocked PI3K signaling and synergized with MEK inhibitors in HRAS-mutated cancer cell lines; covalent KRASG12C inhibitors blocked MEK signaling and synergized with PI3K inhibitors in KRASG12C-mutated cell lines. Synergy between inhibitors of mutant RAS and RAS effectors was dependent on intact RTK/WT RAS signaling and was lost in both RASless or SOSless MEFs. These results should inform the design of clinical trials for patients with RAS-mutated cancers. Citation Format: Nancy Sealover, Bridget Finniff, Robert Kortum. Wild-type RAS signaling is an essential therapeutic target in RAS-mutated cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B033.

Abstract B039: BIO-PROTAC: Application of cell penetrating scFv to treat undruggable KRAS mutant cancer

Abstract Activation of mutations in RAS has been found in high ratio in human cancers, inducing downstream critical effectors corresponding tumorigenesis. In recent years, RAS-RAF-ERK1/2 pathway is known as a crucial target for the anti-cancer drug development because of high prevalence of ERK activation in human cancers. The attempts in direct targeting to activated RAS, which is GTP bound, has been somewhat successful in the treatment of certain type of cancer, however, still beset by the resistance. Our aim is to develop inhibitor that cell-penetrating scFv (single-chain variable fragment) which targets mutated KRAS (mKBscFv; mutated KRAS binding scFv). The Fv fragment is the smallest unit composed of the variable region of heavy chain and light chain in an immunoglobulin molecule, that has a function of antigen-binding activity. The scFv can specifically bind to an antigen, thereby reducing side effects of non-specific binding seen in chemical drugs. However, due to the lack of cell penetrating ability of scFv, a delivery carrier is often required. In this study, the mKBscFv was measured binding affinity toward wild type KRAS and mutated KRAS using surface plasmon resonance (SPR). The scFv has higher affinity to mutated KRAS than wild type KRAS. A cell-penetrating peptide (CPP) was developed for drug delivery and was expressed at the C-terminus of mKBscFv for transferring cells. The mKBscFv-CPP was tagged with His tag to express in CHO cells, which was purified by FPLC. The purity of mKBscFv-CPP was confirmed by SDS PAGE and immunoblotting. The mKBscFv-CPP treated KRAS mutated cancer cells decreased cell viability in a concentration-dependent manner, and decreased expression of mutated GTP-bound KRAS. In addition to the bioactivity of the mKBscFv, it has further advantage as a proteolysis-targeting chimera (PROTAC). The plasmid DNA was prepared by adding a VHL-expressing sequence to the C-terminus of mKBscFv (mKBscFv-Vh). This gene was transfected into KRAS mutated cancer cells to express the protein, and as a result mutated GTP-bound KRAS was reduced. A CPP peptide domain was added to the C-terminus of this gene to express it as a protein in CHO cells. The mutated GTP-bound KRAS was reduced by mKBscFv-Vh-CPP in KRAS mutated cacner cells. Taken together, mKBscFv that binds to mutated KRAS was developed, and it can be expected to become a new protein anticancer drug effective against mutated KRAS-mediated cancer cells by fusing cell penetrating peptide and PROTAC technology. Citation Format: Gookjin Yoon, Beom Soo Jo, Dong Woo Lee, Jinwook Yang, Min-Ho Park, Sanghui Seok, Jue Yeon Lee, Chong Pyoung Chung, Yoon Shin Park, Yoon Jeong Park. BIO-PROTAC: Application of cell penetrating scFv to treat undruggable KRAS mutant cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B039.