Targeting Mutant KRAS Research Articles

Protein-RNA interaction dynamics reveal key regulators of oncogenic KRAS-driven cancers

KRAS is one of the most frequently mutated oncogenes across various cancers. Oncogenic KRAS mutations rewire cellular signaling, leading to significant alterations in gene expression. RNA-binding proteins (RBPs) play a pivotal role in gene expression regulation by post-transcriptionally controlling various aspects of RNA metabolism. It has become clear that interactions between RBPs and RNA are frequently dysregulated in numerous cancers. However, how oncogenic KRAS mutations reshape the post-transcriptional regulatory network mediated by RBPs remains poorly understood. In this study, we systematically dissected oncogenic KRAS-driven alterations of RNA-RBP networks. We identified 35 cancer-associated RBPs with either increased or decreased RNA binding upon oncogenic KRAS activation, including PDCD11, which is essential for the viability of KRAS mutant cancers, and ELAVL2, which regulates cell migration in KRAS mutant lung cancers. Our study serves as a crucial resource for elucidating RBP regulatory networks in KRAS mutant cancers and may provide new avenues for therapeutic strategies targeting KRAS mutant malignancies.

A Novel Lipid Nanoparticle NBF-006 Encapsulating Glutathione S-Transferase P siRNA for the Treatment of KRAS-driven Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers, and KRAS mutations occur in 25-30% of NSCLC. Our approach to developing a therapeutic with the potential to target KRAS mutant NSCLC was to identify a new target involved in modulating signaling proteins in the RAS pathway. Glutathione S-Transferase P (GSTP) known as a Phase II detoxification enzyme has more recently been identified as a modulator of MAP kinase-related cell-signaling pathways. Therefore, developing a GSTP siRNA may be an effective therapeutic approach to treat KRAS mutant NSCLC. The lead drug product candidate (NBF-006) is a proprietary siRNA-based lipid nanoparticle (LNP) comprising GSTP siRNA (NDT-05-1040). Here, studies using a panel of KRAS mutant NSCLC cell lines demonstrated that NDT-05-1040 is a very potent and selective GSTP siRNA inhibitor. Our Western blot analysis showed that NDT-05-1040 effectively decreased the phosphorylation of MAPK and PI3K pathway components while upregulating apoptotic signaling cascade. Our in vivo studies revealed statistically significant higher distribution of NBF-006 to the lungs and tumor as compared to liver. In the subcutaneous and orthotopic tumor models, NBF-006 led to a statistically significant and dose dependent anti-tumor growth inhibition. Further, quantitative image analysis of PCNA and PARP staining showed that NBF-006 decreased proliferation and induced apoptosis, respectively, in tumors. Additionally, in a surgically implanted orthotopic lung tumor model, the survival rate of the NBF-006 treatment group was significantly prolonged (P <0.005) as compared to the vehicle control group. Together, these preclinical studies supported advancement of NBF-006 into clinical studies.

Hope on the Horizon: A Revolution in KRAS Inhibition Is Creating a New Treatment Paradigm for Patients with Pancreatic Cancer.

KRAS is the most frequently altered oncogene in pancreatic ductal adenocarcinoma, in which the aberrantly activated RAS signaling pathway plays pleiotropic roles in tumor initiation and maintenance. Nearly four decades after the discovery of the RAS oncoprotein, a multitude of pharmacologic inhibitors are now available that directly target mutant KRAS. This In Focus commentary, published simultaneously with the 2024 AACR Special Conference on Pancreatic Cancer, summarizes the current state of this rapidly changing field, including preclinical data and emerging clinical trends with respect to therapeutic efficacy, mechanisms of resistance, and potential combinations to maximize clinical benefit from this promising class of therapies.

Novel druggable space in human KRAS G13D discovered using structural bioinformatics and a P-loop targeting monoclonal antibody

KRAS belongs to a family of small GTPases that act as binary switches upstream of several signalling cascades, controlling proliferation and survival of cells. Mutations in KRAS drive oncogenesis, especially in pancreatic, lung, and colorectal cancers (CRC). Although historic attempts at targeting mutant KRAS with small molecule inhibitors have proven challenging, there are recent successes with the G12C, and G12D mutations. However, clinically important RAS mutations such as G12V, G13D, Q61L, and A146T, remain elusive drug targets, and insights to their structural landscape is of critical importance to develop novel, and effective therapeutic concepts. We present a fully open, P-loop exposing conformer of KRAS G13D by X-ray crystallography at 1.4–2.4 Å resolution in Mg2+-free phosphate and malonate buffers. The G13D conformer has the switch-I region displaced in an upright position leaving the catalytic core fully exposed. To prove that this state is druggable, we developed a P-loop-targeting monoclonal antibody (mAb). The mAb displayed high-affinity binding to G13D and was shown using high resolution fluorescence microscopy to be spontaneously taken up by G13D-mutated HCT 116 cells (human CRC derived) by macropinocytosis. The mAb inhibited KRAS signalling in phosphoproteomic and genomic studies. Taken together, the data propose novel druggable space of G13D that is reachable in the cellular context. It is our hope that these findings will stimulate attempts to drug this fully open state G13D conformer using mAbs or other modalities.

The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma.

KRAS gene mutations are common in pancreatic ductal adenocarcinoma (PDAC), but targeting mutant KRAS is still challenging. Here, an endoribonuclease-prepared small interfering RNA (esiRNA) library was used to screen new kinases that play critical roles in PDAC driven by KRAS gene mutations, and serine/threonine kinase 31 (STK31) was identified and characterized as a potential therapeutic target for KRAS-mutant PDAC. Our results showed that STK31 was upregulated in KRAS-mutant PDAC patients with poor survival and highly expressed in PDAC cell lines with KRASG12D mutation. Inhibition of STK31 in KRAS-mutant cell lines significantly reduced PDAC cell growth invitro and hindered tumor growth invivo. Gain and loss of function experiments revealed that STK31 is a downstream target of KRAS in PDAC. A pharmacological inhibition assay showed MAPK/ERK signaling involved in STK31 regulation. The further mechanistic study validated that c-Jun, regulated by KRAS/MAPK signaling, directly modulates the transcription level of STK31 by binding to its promoter region. Through RNA sequencing, we found that the cell cycle regulators CCNB1 and CDC25C are downstream targets of STK31. Taken together, our results indicate that STK31, which is the downstream target of the KRAS/MAPK/ERK/c-Jun signaling pathway in KRAS-mutant PDAC, promotes PDAC cell growth by modulating the expression of the cell cycle regulators CCNB1 and CDC25C.

AFNT-211: A phase 1 study of autologous CD4+ and CD8+ T cells engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR, a CD8α/β coreceptor, and a FAS41BB switch receptor in patients with advanced/metastatic solid tumors.

TPS8650 Background: Activating mutations in KRAS (including KRAS G12V) are well-described oncogenic drivers in solid tumors. Patients with KRAS driver mutations have a poor prognosis, and most KRAS-mutated cancers lack effective therapies. T cell receptor (TCR)-T cell therapies targeting mutant KRAS have demonstrated proof of concept in the clinic, but duration of response remains a challenge. AFNT-211 represents a novel strategy to address the immunosuppressive tumor microenvironment and improve response rate and duration in solid tumors. AFNT-211 autologous CD4+ and CD8+ T cells are engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR; the wildtype CD8α/β coreceptor; and a FAS-41BB switch receptor. The intended mechanism of action is recognition and elimination of KRAS G12V-mutated tumor cells by AFNT-211. AFNT-211 is designed to enhance anti-tumor activity via the following mechanisms of action as shown in preclinical models: (1) the CD8α/β coreceptor enables MHC-I– restricted TCR recognition of the KRAS G12V target by CD4+ T cells; (2) engaging CD4+ helper T cell responses drives enhanced CD8+ T cell cytotoxic activity and prevents T cell exhaustion; (3) the FAS-41BB chimeric switch receptor drives increased T cell activity, circumventing native FAS apoptotic signaling. Methods: This ongoing Phase 1, first-in-human, multicenter, open-label study of AFNT-211 (1) evaluates safety/tolerability, (2) determines the optimal biological dose (OBD) and recommended Phase 2 dose (RP2D), and (3) assesses preliminary antitumor activity. The study consists of 2 parts: dose escalation and dose expansion. Dose escalation is guided by the Bayesian optimal interval Phase 1/2 (BOIN12) design to determine the OBD. The BOIN12 design quantifies the desirability of a dose in terms of toxicity-efficacy tradeoff and adaptively allocates patients to the dose with the highest estimated desirability. The RP2D will be selected based on the BOIN12 design and the totality of benefit/risk assessment. Patients enrolling in dose expansion will be treated at the OBD/RP2D in indication-specific cohorts. This study is conducted in patients aged ≥ 18 years who are HLA-A*11:01-positive with advanced/metastatic solid tumors harboring a KRAS G12V mutation, and intolerance of/progression on at least 1 prior systemic therapy. Patients undergo leukapheresis to collect T cells for the manufacturing of AFNT-211 and receive lymphodepleting chemotherapy prior to AFNT-211 infusion. This is followed by a 28-day dose-limiting toxicity observation period (dose escalation only) and a post-treatment follow-up period for 24 months/ until disease progression. The study is open for recruitment in the United States. Clinical trial information: NCT06105021 .

Abstract CT076: AFNT-211: A phase1 study of autologous CD4+ and CD8+ T cells engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR; CD8α/β coreceptor; and a FAS41BB switch receptor in patients with advanced/metastatic solid tumors

Abstract Background: Activating mutations in KRAS (including KRAS G12V) are well-described oncogenic drivers in solid tumors. Patients with KRAS driver mutations have a poor prognosis, and most KRAS-mutated cancers lack effective therapies. T cell receptor (TCR)-T cell therapies targeting mutant KRAS have demonstrated proof of concept in the clinic, but duration of response remains a challenge. AFNT-211 represents a novel strategy to address the immunosuppressive tumor microenvironment and improve response rate and duration in solid tumors. Rationale: AFNT-211 autologous CD4+ and CD8+ T cells are engineered to express a high avidity HLA-A*11:01restricted, KRAS G12V-specific, transgenic TCR; the wildtype CD8α/β coreceptor; and a FAS-41BB switch receptor. The intended mechanism of action is recognition and elimination of KRAS G12V-mutated tumor cells by AFNT-211. AFNT-211 is designed to enhance anti-tumor activity via the following mechanisms of action as shown in preclinical models: (1) the CD8α/β coreceptor enables MHC-I-‍restricted TCR recognition of the KRAS G12V target by CD4+ T cells; (2) engaging CD4+ helper T cell responses drives enhanced CD8+ T cell cytotoxic activity and prevents T cell exhaustion; (3) the FAS-41BB chimeric switch receptor drives increased T cell activity, circumventing native FAS apoptotic signaling. Methods: This ongoing Phase 1, first-in-human, multicenter, open-label study of AFNT-211 (1) evaluates safety/tolerability, (2) determines the optimal biological dose (OBD) and recommended Phase 2 dose (RP2D), and (3) assesses preliminary antitumor activity. The study consists of 2 parts: dose escalation and dose expansion. Dose escalation is guided by the Bayesian optimal interval Phase 1/2 (BOIN12) design to determine the OBD. The BOIN12 design quantifies the desirability of a dose in terms of toxicity-efficacy tradeoff and adaptively allocates patients to the dose with the highest estimated desirability. The RP2D will be selected based on the BOIN12 design and the totality of benefit/risk assessment. Patients enrolling in dose expansion will be treated at the OBD/RP2D in indication-specific cohorts. This study is conducted in patients aged ≥ 18 years who are HLA-A*11:01-positive with advanced/metastatic solid tumors harboring a KRAS G12V mutation, and intolerance of/progression on at least 1 prior systemic therapy. Patients undergo leukapheresis to collect T cells for the manufacturing of AFNT-211 and receive lymphodepleting chemotherapy prior to AFNT-211 infusion. This is followed by a 28-day dose-limiting toxicity observation period (dose escalation only) and a post-treatment follow-up period for 24 months/until disease progression. The study is open for recruitment in the United States (NCT06105021). Citation Format: Shaunica Mitchell, Binaish Khan, Francis Payumo, E. Gabriela Chiorean, Zhubin Gahvari, J. Randolph Hecht, Michael Hurwitz, Rom Leidner, Heinz-Josef Lenz, Meredith Pelster, Salman Punekar, Adam Schoenfeld, Dan Zhao, Markus Vallaster, Dirk Nagorsen. AFNT-211: A phase1 study of autologous CD4+ and CD8+ T cells engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR; CD8α/β coreceptor; and a FAS41BB switch receptor in patients with advanced/metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT076.

Abstract 1667: The role of CCN1 in mutant K-RAS addiction in pancreatic cancer

Abstract Mutant K-RAS addiction of pancreatic ductal adenocarcinoma cells (PDAC) coordinates their transformation, proliferation, and survival. Therefore, initially, it had an impression that targeting mutant K-RAS could be an ideal strategy to treat PDAC. However, after decades of studies, no effective RAS inhibitors reached the clinic, indicating that RAS-oncoprotein is an undruggable target in PDAC. Thus, identifying a new target linked with K-RAS signaling was most needed. Using genetically engineered cell lines and K-RAS-driven genetically engineered mouse models (KC or KPC) with or without pancreas-specific CCN1 conditional knockout background, we identified a positive feedback circuit between CCN1 and mutant K-RAS expression. Moreover, blocking this feedback via conditional knockout of CCN1 in the pancreas significantly reduced invasive phenotypes, PDAC growth, and metastasis in the lung in KC and KPC mice. Mechanistically, we found that the K-RAS-CCN1 feedback circuit is regulated by microRNA miR-145 via a feedforward mechanism. Collectively, these studies highlight the clinical potential of targeting CCN1 in PDAC. (VA Merit Grant supports this work) Citation Format: Anjali Kambhampati, Archana De, Inamul Haque, Snigdha Banerjee, Sushanta K. Banerjee. The role of CCN1 in mutant K-RAS addiction in pancreatic cancer [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 1667.

Abstract 6501: Development and characterization of covalent inhibitors of the RAS-PIK3CA interaction

Abstract RAS proteins are membrane bound GTPases that when hyperactivated, act as oncogenes through activation of the MAPK &amp; PI3K pathways. Each of these pathways has oncogenic potential, as exemplified by the high frequency of BRAF mutations in melanoma and PIK3CA and PTEN mutations in breast and gynecological cancers. Simultaneous activation of these pathways, as occurs in RAS driven cancers, generates aggressive cancers that present a significant clinical challenge. KRAS, the most commonly mutated RAS isoform, is also the most frequently mutated oncogene in cancer. While treatment options have improved for a subset of these patients due to the accelerated approval of KRAS-G12C inhibitors, the rapid development of resistance highlights the continued need for effective treatments. In RAS driven cell and animal models, dual inhibition of the MAPK &amp;PI3K pathways has shown superior efficacy relative to targeting the individual pathways, however dose limiting toxicities in humans have prevented this combination strategy from finding clinical success. While physiological activation of the MAPK pathway is RAS dependent, the interaction between RAS and the catalytic subunit of PI3Kα, PIK3CA, serves as an amplifier but not a primary activator of this pathway. This interaction is particularly important in cancerous cells as it serves to amplify basal PI3K activity and support tumor progression. Conversely, in healthy cells, RAS independent activation of PI3K by upstream signaling factors is sufficient for maintaining physiological homeostasis. Unfortunately, traditional strategies of targeting the PI3K pathway have been unable to capitalize on this therapeutic opportunity as they do not discriminate between RAS dependent and RAS independent signaling. This leads to on-target dose-limiting toxicities, most commonly hyperglycemia and rash. We have discovered small molecules that disrupt the RAS:PIK3CA interaction through covalent ligation of C242, in the RAS binding domain, adjacent to the RAS binding interface. Using a Nanobit system to measure the RAS:PIK3CA interaction and signaling assays in “RAS active” cells, we have optimized molecules that disrupt the RAS:PIK3CA interaction and inhibit RAS mediated activation of PIK3CA. These molecules show robust inhibition of PIK3CA signaling, and tumor growth inhibition in cancer models, but unlike PIK3CA active site inhibitors, they do not impact glucose handling. Profound efficacy is seen when these RAS:PIK3CA inhibitors are used in combination with an agent targeting the MAPK pathway or with a therapy directly targeting mutant KRAS. Finally, we found that ligation of C242 on PIK3CA blocks HER2/3 driven activation of PI3Kα in a RAS independent manner. Overall, our data supports the clinical investigation of these molecules, particularly in combination with rationally chosen therapies where they may provide a tolerable and efficacious means of blocking the PI3K pathway. Citation Format: Joseph Klebba, Junko Tamiya, Jinwei Wang, Hui Miao, Kelsey Lamb, Aaron Snead, Antonio Esparza-Oros, Richard Lin, Chanyu Yue, Stephanie Grabow, Steffen Bernard, Yongsheng Liu, Benjamin Horning, Melissa Hoffman, Marie Pariollaud, Taylor Wyrick, Holly Parker, John Sigler, Martha Pastuszka, David Weinstein, Todd Kinsella, Matt Patricelli. Development and characterization of covalent inhibitors of the RAS-PIK3CA interaction [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 6501.

Abstract 6717: Targeting mutant KRAS proteins with novel TCR-mimic fully human antibodies

Abstract Mutated KRAS proteins are ideal cancer targets, as they are expressed frequently and specifically in certain solid tumors. A large proportion of human colorectal cancer and pancreatic ductal adenocarcinoma express the tumor driver KRAS gene mutations G12V/G12D, but drugs targeting G12V/G12D are not available, revealing a huge unmet clinical need. While small molecules often fail to target the KRAS mutation G12V/G12D, T cell receptor-mimic (TCR-mimic) antibodies can specifically recognize KRAS mutations presented by human leukocyte antigen (HLA), opening up possibilities for targeting such intracellular antigens. Here, we discovered novel antibodies highly specific to G12V/HLA and G12D/HLA complexes by immunizing our proprietary RenTCR-mimicTM mice and using high-throughput Beacon-based screening. These TCR-mimic antibodies have higher affinities compared to endogenous TCRs, which may effectively reduce the possibility of tumor escape. Germline distribution analysis indicated their high sequence diversity, which suggests diverse epitope targeting. Although pancreatic cancer is extremely difficult to treat and has an extremely low KRAS mutant peptide-HLA complex density on the cell surface, our TCR-mimic antibodies exhibited potent in vitro tumor lysis activity when assembled into CD3 T cell engagers. Furthermore, these antibodies demonstrated convincing off-target safety. Together, our results indicate promising therapeutic potential of these KRAS mutation-targeted TCR-mimic antibodies for the treatment of solid tumors. Citation Format: Jun Du, Wanbo Tang, Xin Jiao, Limin Zhao, Pengfei Du, Yuqi Zhang, Jian Bao, Han Chen, Chaoshe Guo, W. Frank An. Targeting mutant KRAS proteins with novel TCR-mimic fully human antibodies [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 6717.

Myotubularin-related-protein-7 inhibits mutant (G12V) K-RAS by direct interaction

Inhibition of K-RAS effectors like B-RAF or MEK1/2 is accompanied by treatment resistance in cancer patients via re-activation of PI3K and Wnt signaling. We hypothesized that myotubularin-related-protein-7 (MTMR7), which inhibits PI3K and ERK1/2 signaling downstream of RAS, directly targets RAS and thereby prevents resistance. Using cell and structural biology combined with animal studies, we show that MTMR7 binds and inhibits RAS at cellular membranes. Overexpression of MTMR7 reduced RAS GTPase activities and protein levels, ERK1/2 phosphorylation, c-FOS transcription and cancer cell proliferation in vitro. We located the RAS-inhibitory activity of MTMR7 to its charged coiled coil (CC) region and demonstrate direct interaction with the gastrointestinal cancer-relevant K-RASG12V mutant, favouring its GDP-bound state. In mouse models of gastric and intestinal cancer, a cell-permeable MTMR7-CC mimicry peptide decreased tumour growth, Ki67 proliferation index and ERK1/2 nuclear positivity. Thus, MTMR7 mimicry peptide(s) could provide a novel strategy for targeting mutant K-RAS in cancers.

Nucleic Acid-Based Approaches to Tackle KRAS Mutant Cancers

Activating mutations in KRAS are highly relevant to various cancers, driving persistent efforts toward the development of drugs that can effectively inhibit KRAS activity. Previously, KRAS was considered ‘undruggable’; however, the recent advances in our understanding of RNA and nucleic acid chemistry and delivery formulations have sparked a paradigm shift in the approach to KRAS inhibition. We are currently witnessing a large wave of next-generation drugs for KRAS mutant cancers—nucleic acid-based therapeutics. In this review, we discuss the current progress in targeting KRAS mutant tumors and outline significant developments in nucleic acid-based strategies. We delve into their mechanisms of action, address existing challenges, and offer insights into the current clinical trial status of these approaches. We aim to provide a thorough understanding of the potential of nucleic acid-based strategies in the field of KRAS mutant cancer therapeutics.

Targeting Mutant KRAS with Novel Mutant-Specific Pharmacological Inhibitors: Activity Against Myeloma and Other Lymphoid Malignant Cells and Molecular Mechanisms of Resistance

Oncogenic RAS mutations are frequent genomic drivers in hematologic neoplasias such as multiple myeloma (MM) but are traditionally considered non-“druggable”. Novel specific inhibitors of G12C and G12D KRAS mutants are clinically active in solid tumors, but their impact on KRAS-mutant lymphoid malignancies has not been explored. Here, we examined the activity of these inhibitors against G12C/D KRAS-mutant cells from MM, B-ALL and T-ALL; the functional sequelae of pharmacological KRAS inhibition; and the potential mechanisms of resistance to these agents. We assessed the activity of novel G12C- (MRTX1257) and G12D- (MRTX1133) specific pharmacological KRAS inhibitors against 2 MM lines with G12C (KHM1B, XG7) and 2 with G12D (KARPAS620, KP6) KRAS mutations; as well as the G12D mutant KOPN-8 (B-ALL) and CCRF-CEM (T-ALL) lines. KRAS inhibitors were tested at concentration ranges achievable in patients with solid tumors. Potent and mutant-specific activity was achieved with both of these KRAS inhibitors in their respective lines, with IC50 in the range of 5-150 nM after 3-7d treatment. This level of in vitro activity was similar to KRAS-mutant lines (e.g., in our in vitro studies and also in the literature) from solid tumors that are clinically responsive to these agents. Furthermore, the KRAS inhibitors effectively abrogated downstream MAPK and AKT pathway activation (e.g. phosphorylation of ERK or AKT by immunoblotting). MRTX1133 led to potent induction of cell death of KARPAS620, KP6 and KOPN8 cells (flow cytometry for Annexin V/PI staining) while MRTX1257 induced cell death in KHM1B and had a predominantly cytostatic effect on XG7 cells. Combinations of MRTX1257 with clinically established anti-MM drugs (incl. melphalan, bortezomib, pomalidomide, trametinib) caused in some cases supra-additive effects, but - more importantly - showed no antagonism with any combination. RNA sequencing revealed that KRAS inhibitor treatment downregulated RAS effector genes (e.g., ETV4, ETV5), and negative regulators of RAS signaling (e.g., DUSP6, SPRY4 reflecting a RAS-driven negative feedback loop controlling expression of these genes), and molecules involved in cell cycle regulation (e.g., D-type cyclin), anti-apoptosis, and DNA replication/repair. Conversely, KRAS inhibition upregulated genes involved in cell cycle arrest and stress responses. Importantly, we performed genome-scale CRISPR activation studies in 4 MM lines with G12C/G12D mutant KRAS to identify genes whose gain-of-function (GOF) enhances or suppresses MM cell response to the respective KRAS inhibitors. Top “hits” from these screens were validated with individual sgRNAs. CRISPR activation of mutant KRAS itself promotes MM cell escape from treatment, likely due to stoichiometric reasons and decreased target saturation at a given drug concentration. Notably, CRISPR activation of positive regulators of RAS-MAPK signaling (e.g., SHOC2), and upstream surface receptors (e.g., EGFR) were among top genes mediating KRAS inhibitor resistance. Beyond KRAS itself, these top “hit” genes are typically not recurrently mutated, amplified, differentially expressed (e.g., compared with normal plasma cells) in MM and are not associated with “high-risk” clinical behavior. Some (e.g., EGFR) of the genes identified from these CRISPR studies are not typically expressed in MM cells at diagnosis, thus these results outline how ectopic expression (e.g., EGFR) or overexpression of these “hit” genes could represent candidate biomarkers for decreased response or early relapse in future clinical studies of KRAS inhibitors in KRAS-mutant MM. Ongoing studies are addressing how these genomic perturbations may affect the in vivo response of KRAS-mutant cells to KRAS inhibitors alone or in combinations with established therapies. Our study documents that novel KRAS G12C and G12D inhibitors exhibit potent and mutant-specific activity against MM and other lymphoid malignant cells at dose levels that are clinically achievable in solid tumor patients. This study also provides functional evidence about genes and molecular pathways that regulate MM cell sensitivity vs. resistance to KRAS inhibitors. These results create a framework for ongoing and future efforts to translate the use of KRAS inhibitors into clinical studies for MM and other lymphoid malignancies.

Target Mutant KRAS with Molecular Glue

Target Mutant KRAS with Molecular Glue

Modulation of the proteostasis network promotes tumor resistance to oncogenic KRAS inhibitors.

Despite substantial advances in targeting mutant KRAS, tumor resistance to KRAS inhibitors (KRASi) remains a major barrier to progress. Here, we report proteostasis reprogramming as a key convergence point of multiple KRASi-resistance mechanisms. Inactivation of oncogenic KRAS down-regulated both the heat shock response and the inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response, causing severe proteostasis disturbances. However, IRE1α was selectively reactivated in an ER stress-independent manner in acquired KRASi-resistant tumors, restoring proteostasis. Oncogenic KRAS promoted IRE1α protein stability through extracellular signal-regulated kinase (ERK)-dependent phosphorylation of IRE1α, leading to IRE1α disassociation from 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) E3-ligase. In KRASi-resistant tumors, both reactivated ERK and hyperactivated AKT restored IRE1α phosphorylation and stability. Suppression of IRE1α overcame resistance to KRASi. This study reveals a druggable mechanism that leads to proteostasis reprogramming and facilitates KRASi resistance.

Targeting TEAD-ious resistance

Recent advances in targeting mutant KRAS are limited by resistance. A recent study in Nature Cancer by Hagenbeek et al. utilizes a novel inhibitor that targets the TEAD transcription factor, GNE-7883, to overcome resistance to KRAS inhibitors. Thus, TEAD inhibitors may maximize the durability of KRAS inhibitors in patients with cancer.

Translational relevance of SOS1 targeting for KRAS-mutant colorectal cancer.

It has been challenging to target mutant KRAS (mKRAS) in colorectal cancer (CRC) and other malignancies. Recent efforts have focused on developing inhibitors blocking molecules essential for KRAS activity. In this regard, SOS1 inhibition has arisen as an attractive approach for mKRAS CRC given its essential role as a guanine nucleotide exchange factor for this GTPase. Here, we demonstrated the translational value of SOS1 blockade in mKRAS CRC. We used CRC patient-derived organoids (PDOs) as preclinical models to evaluate their sensitivity to SOS1 inhibitor BI3406. A combination of in silico analyses and wet lab techniques was utilized to define potential predictive markers for SOS1 sensitivity and potential mechanisms of resistance in CRC. RNA-seq analysis of CRC PDOs revealed two groups of CRC PDOs with differential sensitivities to SOS1 inhibitor BI3406. The resistant group was enriched in gene sets involving cholesterol homeostasis, epithelial-mesenchymal transition, and TNF-α/NFκB signaling. Expression analysis identified a significant correlation between SOS1 and SOS2 mRNA levels (Spearman's ρ 0.56, p < 0.001). SOS1/2 protein expression was universally present with heterogeneous patterns in CRC cells but only minimal to none in surrounding nonmalignant cells. Only SOS1 protein expression was associated with worse survival in patients with RAS/RAF mutant CRC (p = 0.04). We also found that SOS1/SOS2 protein expression ratio >1 by immunohistochemistry (p = 0.03) instead of KRAS mutation (p = 1) was a better predictive marker to BI3406 sensitivity of CRC PDOs, concordant with the significant positive correlation between SOS1/SOS2 protein expression ratio and SOS1 dependency. Finally, we showed that GTP-bound RAS level underwent rebound even in BI3406-sensitive PDOs with no change of KRAS downstream effector genes, thus suggesting upregulation of guanine nucleotide exchange factor as potential cellular adaptation mechanisms to SOS1 inhibition. Taken together, our results show that high SOS1/SOS2 protein expression ratio predicts sensitivity to SOS1 inhibition and support further clinical development of SOS1-targeting agents in CRC.

Abstract 6122: Targeting mutant p53 and KRAS for novel pancreatic cancer therapy by combination treatment of simvastatin and imipridones

Abstract KRAS mutation is found in 95% pancreatic ductal adenocarcinoma (PDAC). TP53 is altered in 70% of patients with PDAC that co-occur with KRAS mutations. Enhanced de novo cholesterol biosynthesis is a hallmark of cancer cells. P53 inhibits the mevalonate pathway to mediate tumor repression. p53 mutations drive de novo cholesterol pathway activation and are required for the proliferation of KRAS-mutant cancers. Inhibition of the mevalonate pathway leads to feedback activation of SREBP-2, which restores mevalonate pathway and leads to re-activation of KRAS and restoration of mutant p53 GOF. Imipridones induced ATF4 through the action of mitochondria protease ClpP. Simvastatin triggers ATF4 activation through inhibition of mevalonate pathway. We hypothesize that combined treatment with imipridones can overcome the resistance to simvastatin by two different mechanisms: (1) Imipridones inhibit oxidative phosphorylation through ClpP hyperactivation and result in AMPK activation. AMPK phosphorylates and inhibits SREBP-2 transcriptional function, which blocks the feedback activation of SREBP-2. (2) Enhanced ATF4 and CHOP induction through different pathways leads to cancer cell death. We also expect that the combined treatment through sustained induction of ATF4 and CHOP can sensitize cancer cells to imipridone treatment. Mouse and human PDAC cell lines (KPCY, HPAF-II, and PANC-1) with p53 and KRAS mutations were used to test the effects of combination treatment of simvastatin and imipridones. The combined treatments synergistically inhibited the proliferation of p53 and KRAS co-mutant cell lines. For example, the synergy scores of simvastatin and ONC201 combination are 75 (KPCY), 15 (HPAF-II), and 14 (PANC-1). Enhanced cell death is consistent with sustained and enhanced induction of ATF4 and CHOP compared to single drug treatment alone. Imipridones activated AMPK in the tested PDAC cell lines. Imipridones potently inhibit AKT and ERK1/2 activation at 72-hour time point, which might also contribute to block above mentioned feedback activation of KRAS. We also observed that cell lines with a structural mutant of p53 are more sensitive to the combination treatments than a cell line with a DNA-contact mutant of p53, which is consistent with the fact that simvastatin treatment leads to degradation of structural p53 mutants. Surprisingly, our data indicated that simvastatin potently inhibits kinase WEE1 and leads to CDK1 dephosphorylation and activation, which has not been reported before. Together, our data is consistent with publications that p53 mutated cancer cell lines are sensitive to statin treatments. In short, combination treatment may overcome simvastatin resistance and/or imipridone resistance by modulating ATF4/CHOP and SREBP-2 activity. Citation Format: Xiaobing Tian, Wafik S. Deiry. Targeting mutant p53 and KRAS for novel pancreatic cancer therapy by combination treatment of simvastatin and imipridones [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 6122.

Abstract 2218: The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma

Abstract KRAS is frequently mutated in pancreatic ductal adenocarcinoma (PDAC). However, targeting mutant KRAS is still challenging. Kinase inhibitors are ideal targeted therapeutics for mutant KRAS-driven cancer. In our previous study, an esiRNA screening was prepared to identify some vital kinases that took part in KRAS mutant-driven pancreatic cancer. The STK31 was identified as a potential therapeutic target in KRAS mutant PDAC. This study aimed to investigate the underlying mechanism of STK31 in KRAS mutant PDAC as well as its therapeutic potential. The results demonstrated that STK31 was upregulated in KRAS mutant PDAC patients with poor survival. STK31 was also highly expressed in PDAC cell lines with KRAS mutant background. Inhibition of STK31 in KRAS mutant cell lines significantly reduced PDAC cell proliferation, colony formation, and migration. Gain and loss of function experiments revealed that STK31 was a downstream target of KRAS in PDAC. Pharmacological inhibition of several signaling pathways involved in KRAS showed MAPK/ERK signaling involved in STK31 regulation. Then, further mechanistic study validated that c-Jun, regulated by KRAS/MAPK/ERK signaling, directly regulated the transcription level of STK31 by binding to its promoter region. In summary, our results show that STK31 promotes PDAC cell proliferation under the control of the KRAS/MAPK/ERK/c-Jun transcriptional axis in PDAC. STK31 may act as a potential target in PDAC treatment. Citation Format: Yuting Liu, Shing Chun Tang, Yangchao Chen. The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma [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 2218.

Abstract 4043: Stimulation of the mapk and pi3k/akt pathways in pancreatic cancer cells by polyisoprenylated cysteinyl amide inhibitors

Abstract Pancreatic cancer is the third deadliest cancer with a 5-year survival rate of less than 10%. Over 90% of reported cases are driven by KRAS mutations. KRAS signals through the MAPK and PI3K pathways. Targeting mutant KRAS has been a decades-long challenge until recently whereby drugs that react with specific mutant proteins have been developed. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) are potential anticancer agents with an alternative design strategy targeting polyisoprenylation-dependent processes of hyperactive mutant or overexpressed G-proteins. The effect of PCAIs on the viability and downstream mediators of KRAS signaling was determined on PANC-1 and MIAPaCa-2 cells. Of the 16 different analogs tested, NSL-YHJ-2-45 and NSL-YHJ-2-27 were the most potent with EC50 values of 3.6 and 3.8 against PANC-1 cells, respectively. For MIAPaCa-2 cells, NSL-YHJ-2-27 was the most potent with an EC50 value of 2.2. Western blotting analysis of PCAIs-treated PANC-1 cells revealed that phosphorylated BRAF, MEK 1/2, ERK 1/2 and p90RSK increased by 64, 129, 152 and 79%, respectively, while phosphorylated CRAF levels decreased by 27%. Moreover, monomeric G-proteins, CDC42, RHOA and RAC 1/2/3 protein levels decreased by 20, 58 and 13%, respectively. NSL-YHJ-2-27 (5 µM) also increased the levels of pAKT (Ser 473) and pAKT (Thr 308) by 72 and 192%, respectively. When MIAPaCa-2 cells were treated with 0-5 µM NSL-YHJ-2-27, MEK 1/2, ERK 1/2 and p90RSK phosphorylation increased by 78, 270 and 260%, respectively. However, at 5 µM NSL-YHJ-2-27, there were no significant changes in protein levels of the monomeric G proteins RAC 1/2/3 and RHOA, but protein levels of CDC42 increased by 132%. NSL-YHJ-2-27 (5 µM) increased the levels of pAKT (Ser 473) and pAKT (Thr 308) of MIAPaCa-2 cells by 97 and 82%, respectively. The results indicate the effect of PCAIs on the phosphorylation of important kinases involved in MAPK and AKT pathways. These effects on these signaling pathways strongly indicate the potential of the PCAIs against RAS-driven cancers. Citation Format: Kweku Ofosu-Asante, Jassy M. Lazarte, Amarendar G. Burra, Nazarius S. Lamango. Stimulation of the mapk and pi3k/akt pathways in pancreatic cancer cells by polyisoprenylated cysteinyl amide inhibitors. [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 4043.