KRAS G12D Research Articles

Abstract A094: Preclinical characterization of HMPL-415, a second-generation SHP2 inhibitor

Abstract Background: Oncogenic activation of the RAS/MAPK signaling pathway is one of the leading causes for driving a variety of cancers. Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) functions downstream of multiple RTKs, and integrates growth factor signals to promote RAS activation, making it an attractive drug target for cancers harboring oncogenic alterations in RAS/MAPK pathway. Herein, we present the preclinical characterization of HMPL-415, a highly potent, selective and non-competitive SHP2 inhibitor, discovered and being currently developed in phase I clinical trial (NCT05886374) by HUTCHMED. Methods: The inhibition of HMPL-415 on the full length and catalytic domain of SHP2 was detected by a biochemical DIFMUP pseudosubstracte-fluorgenic assay. For selectivity, HMPL-415 was assessed against a panel of 413 kinases and 21 phosphatases by Eurofins. The modulation of HMPL-415 on RAS/MAPK signaling cascades was evaluated by western blot. In vitro anti-proliferation activity was measured by CellTiter-Glo luminescent assay. Multiple human xenograft tumors with RAS/MAPK pathway activation were applied in immune-deficient mice to investigate the anti-tumor activity of HMPL-415. Results: In vitro, HMPL-415 displayed higher potency in enzyme (5 fold) and cellular target inhibition (> 20 fold) compared to first-generation SHP inhibitor TNO155. It strongly inhibited full length of human SHP2 with an IC50 of 0.4 nM, while had no inhibitory activity up to 10 µM against the catalytic domain of SHP2. In NCI-H358 (KrasG12C), NCI-H508 (class Ⅲ BRAFG596R), and NCI-H1838 (NF1N184fs) cells, HMPL-415 inhibited p-ERK with IC50s of 1~3 nM, and significantly suppressed downstream molecules p-RSK and p-S6. Through inhibition of RAS/MAPK signaling pathway, HMPL-415 robustly inhibited cell proliferation in a panel of human cancer cell lines with KRAS G12C/V, BRAF class Ⅲ, NF1 loss of function (LOF), RTK mutations or KRAS amplification (median GI50 =1 nM) compared to cell lines harboring KRAS G12D, G13 or Q61 mutations (median GI50 >1 µM), indicating a dependence on specific KRAS nucleotide-cycling for anti-tumor effect. Furthermore, the selectivity across panels of 413 kinases and 21 phosphatases revealed that HMPL-415 should have a low off-target risk. In vivo, oral administration of HMPL-415 showed prolonged and high tumor exposure and sustained inhibition on RAS/MAPK signaling after repeat dosing. In human xenograft models carrying KRAS, BRAF class Ⅲ, NF1LOF, and EGFR alterations, HMPL-415 dose-dependently induced tumor growth inhibition at continuous daily doses that were well tolerated. Tumor regression was observed in most tumor models tested at the dose of 3 mg/kg/day, which demonstrated much superior anti-tumor activity than that of TNO155 at 30 mg/kg/day. Conclusion: HMPL-415 is a highly potent, selective and non-competitive SHP2 inhibitor that demonstrates strong anti-tumor activity in vitro and in vivo, supporting further clinical evaluation. Citation Format: Jia Hu, Jun Ni, Zhihu Gao, Xiaoqing Liu, Hui Zhang, Pan Wang, Min Cheng, Guanglin Wang, Zeyu Zhong, Jian Wang, Yang Sai, Na Yang, Weiguo Qing, Yongxin Ren, Michael Shi, Weiguo Su. Preclinical characterization of HMPL-415, a second-generation SHP2 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A094.

A case description of pulmonary enteric adenocarcinoma with KRAS G12D mutation.

A case description of pulmonary enteric adenocarcinoma with KRAS G12D mutation.

Abstract A127: Identification of therapeutic drug combinations targeting KRAS

Abstract KRAS is the most frequently mutated oncogene and increased development efforts targeting KRAS mutations have yielded recent FDA approvals of two KRAS G12C inhibitors. However, for some indications, there are other KRAS mutations more common than G12C. For Pancreatic ductal adenocarcinoma (comprising more than 90% of pancreatic cancer cases), 94% harbor KRAS mutations, where G12D mutations account for around 45% while G12C mutations are rare (1%). Hence, there is a need to develop methods to target a broader range of KRAS mutations. Beyond direct gene mutation, activation of oncogenic KRAS and downstream MAPK signaling through other genomic alterations may benefit from KRAS-specific therapeutic strategies, especially combination therapy. We have developed a platform, Optim.AI™, that rationally converges upon optimal drug combinations within a defined drug search space, without any prior knowledge of the background of the models. In this study, we utilized the platform to identify efficacious drug combinations that contain a histone deacetylase 4/6 inhibitor, KYAN-001 in the context of KRAS mutations. 7 cell lines (6 prostate and pancreatic cancer with normal, epithelial THLE-2 cell line) were evaluated with Optim.AI. The cancer cells and THLE-2 were seeded into 384-well plates and treated with 155 different combinations consisting of indication-specific drugs the following day. Post-drug treatment cell viability was used for Optim.AI™ analysis, which maps experimental data points to a second-order quadratic function to predict and rank cancer cell killing efficacies for all other permutations of drug combinations. In androgen resistant prostate cancer, Optim.AI identified synergistic interaction between KYAN-001 and a MEK inhibitor. With MEK being downstream of the KRAS pathway, we wanted to evaluate the efficacy of this combination on pancreatic cancer. Interestingly, this combination was demonstrated to be top ranked against PANC-1 and AsPC-1 cell lines, both harboring G12D KRAS mutation, as compared to a cell line with wild-type KRAS, BxPC-3. This was further validated via half-maximal inhibitory curves (IC50), where synergism was also observed for the combination on AsPC-1, but not BxPC-3. KYAN-001 was also shown to exhibit monotherapy sensitivity against pancreatic cancer patient-derived organoids (PDOs), with a mean of 4 µM. This study has successfully demonstrated the ability of Optim.AI™ to identify novel, optimal drug combinations for both prostate and pancreatic cancers, without any prior background knowledge on these cell lines. With MEK being downstream of the KRAS pathway and 94% of those diagnosed with pancreatic cancer have KRAS mutations, the differential sensitivities of this combination across the cell lines presents the utility of the platform in precision medicine, especially for undruggable targets like KRAS. With promising and demonstrated monotherapy efficacy of KYAN-001 on PDOs, further experiments could be done to evaluate the clinical relevance of both KYAN-001 and MEK inhibition on other relevant ex vivo models. Citation Format: Masturah Mohd Abdul Rashid, Lisa Chow, Edward Kai-Hua Chow. Identification of therapeutic drug combinations targeting KRAS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A127.

Metastatic Mullerian adenocarcinoma involving the pancreas initially misdiagnosed as an intraductal papillary mucinous neoplasm (IPMN): A case report, and lessons learned

Abstract Introduction/Objective Metastases to the pancreas from Mullerian primaries are rare and can be confused with primary pancreatic malignancies. Methods/Case Report A 49-year-old woman presented with right lower abdominal pain. She denied any prior history of malignancy but had undergone hysterectomy and bilateral salpingo-oophorectomy for unknown reasons. CT scan revealed an 8.9 cm predominantly cystic mass in the pancreas, highly suspicious of an invasive mucinous neoplasm. EUS-guided FNA of the pancreas showed large, complex papillary fronds of mucinous epithelial cells in a background of abundant extracellular mucin, findings that were interpreted as an intraductal papillary mucinous neoplasm (IPMN) with foci of high-grade dysplasia and possible adenocarcinoma. A subsequent PET/CT scan demonstrated a 3.9 cm hepatic mass and a 3.8 cm perihepatic lesion. FNA of the liver was interpreted as metastatic pancreatic adenocarcinoma. Despite aggressive treatment, her disease progressed. Molecular profiling of her liver tumor demonstrated a pathogenic KRAS G12D mutation and a PDGFRA variant of uncertain significance. Genomic (DNA) and transcriptomic (RNA) analysis indicated a 96% probability that the patient had ovarian or fallopian tube adenocarcinoma and a 4% probability of uterine endometrial adenocarcinoma rather than metastatic pancreatic adenocarcinoma. A panel of immunocytochemical stains was performed on the pancreas and liver FNAs. The tumor cells were strongly positive for CK7, CK19, ER, and WT1, focally positive for PAX8 (nuclear) and P53 (wild-type), and negative for CK20, CDX2, and SATB2. Results (if a Case Study enter NA) NA Conclusion These findings corroborated the molecular analysis suggesting that the patient had metastatic Mullerian adenocarcinoma rather than metastatic pancreatic adenocarcinoma. Her oncologic treatment was adjusted accordingly.

RAS MULTI(ON) Inhibitor RMC-7977 Targets Oncogenic RAS Mutations and Overcomes RAS/MAPK-Mediated Resistance to FLT3 Inhibitors in AML Models

FLT3 inhibitors (FLT3i) such as gilteritinib are clinically active in AML, but their use is limited by resistance due to emergence of clones with RAS/MAPK mutations, both alone and in combination with FLT3 mutations, or with other drivers such as KIT mutations. RAS mutations are also commonly associated with relapse on IDH1/2 inhibitors and the BCL2 inhibitor venetoclax. Importantly, multiple heterogeneous resistant clones are identified at the time of relapse. In addition, 5-10% of all de novo patients harbor oncogenic RAS mutations. Patients with RAS mutations or other mutations that activate RAS/MAPK signaling do not benefit from clinically approved targeted therapies. Targeting oncogenic RAS has been historically challenging and inhibition of downstream effectors of the MAPK pathway, such as MEK, demonstrated modest activity and high toxicity in clinical trials. Effective inhibition of the RAS/MAPK pathway is therefore a critical unmet need in AML. RMC-7977 is a potent, oral small molecule inhibitor of both wild-type and mutant GTP-bound RAS oncoproteins (RAS MULTI) and is a preclinical tool compound representative of the clinical candidate RMC-6236, currently in clinical evaluation (NCT05379985). RMC-7977 non-covalently binds to the intracellular chaperone cyclophilin A, generating a neomorphic interface with high affinity for all isoforms of RAS. The resulting tri-complexes sterically block RAS-effector interactions required for propagating oncogenic signals. We report in vitro data supporting preclinical utility of RAS MULTI(ON) inhibition in AML models harboring RAS mutations, including those with resistance to FLT3i due to hyperactive RAS signaling. RMC-7977 suppressed cell proliferation in AML cell lines driven by FLT3-ITD (Molm-14, MV4-11), KIT N822K (Kasumi-1, SKNO-1) and RAS mutations ( NRAS Q61L -OCIAML-3, HL-60, KRAS G13D - NOMO-1) with IC 50 values between 5 and 33 nM and repressed phosphorylation of downstream effectors of the MAPK pathway MEK, ERK and RSK. We also assessed the activity of RMC-7977 in resistant Molm-14 cells that developed secondary NRAS G12C or NRAS Q61K mutations after long-term exposure to FLT3i. At concentrations as low as 5 nM, RMC-7977 restored sensitivity to gilteritinib in both NRAS-mutant resistant cell lines. Using a caspase3/7 assay, we observed that RMC-7977 induced apoptosis, but to different extents across AML cell lines. We have previously demonstrated that effective MAPK signaling inhibition increases apoptotic dependency on BCL2. Consequently, the addition of venetoclax to RMC-7977 significantly enhanced caspase activation in cell lines with FLT3, KIT and NRAS mutations. In a cell viability assay, RMC-7977 and venetoclax showed high synergistic activity as assessed by Bliss independence model. Given that intrapatient tumor heterogeneity is associated with clinical resistance to targeted therapies, we investigated the in vitro activity of RMC-7977 in models that recapitulate patterns of clonal outgrowth observed in patients treated with FLT3i. We mixed fluorescently-tagged cell lines with FLT3-ITD (Molm-14), FLT3-ITD and NRAS co-mutations (Molm-14 NRAS Q61K) and NRAS-only mutations (OCIAML-3), treated the mixtures with RMC-7977 alone and in combination (with gilteritinib or venetoclax) for 96 hours, then assessed cell viability via flow cytometry. Gilteritinib and the gilteritinib/venetoclax combination selected for survival of cells harboring NRAS mutations, but RMC-7977 inhibited outgrowth of all cell populations. The combination of RMC-7977 and gilteritinib had superior activity in mixtures containing FLT3 and FLT3- NRAS co-mutations, but no additional benefit over RMC-7977 monotherapy in cells harboring NRAS mutations alone. Strikingly, the combination of RMC-7977 and venetoclax potently suppressed cell viability equally in all cell line models and was significantly superior to RMC-7977 alone. In vivo studies investigating the tolerability and activity of RMC-7977 and RMC-7977 combinations in RAS mutant/FLT3i-resistant patient-derived xenograft models are ongoing and will be presented. Collectively, our data provide preclinical evidence that combination therapies leveraging RAS MULTI(ON) inhibition are effective in suppressing RAS-mutant AML clones, a common mechanism of resistance to currently approved targeted therapies in AML and a current area of high unmet clinical need.

N/KRAS-Mutant AML LSCs Originate from Committed Myelomonocytic Progenitors and Drive Clinical Resistance to Venetoclax

Driver mutations in acute myeloid leukemia (AML) often exhibit distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in AML, upon progression or relapsed/refractory disease, in contrast to their function as early drivers in solid tumors. We developed synthetic leukemogenesis models in human induced pluripotent stem cell (iPSC)- and primary cord blood (CB)-derived human hematopoietic stem/progenitor cells (HSPCs) by introducing the prototypical NRAS G12D mutation alone or with other mutational combinations, using CRISPR, and engraftment of a transplantable leukemia into NSG mice as the readout. RAS mutations alone were not sufficient for leukemogenesis, but required specific cooperating mutations. Subsequently, by creating temporally controlled engineered models, we found that RAS mutations are obligatory late events that can only drive leukemic transformation when acquired after, but not before, cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed granulocyte-monocyte progenitors (GMPs) harboring previously acquired driver mutations, through sorting and transplantation and multi-omics experiments. In contrast, acquisition of RAS mutations in earlier progenitors (common myeloid progenitors, CMP), with or without cooperating mutations, blocked the emergence of GMPs and abolished engraftment or gave rise to a myeloproliferative neoplasm. These results indicate that advanced RAS-mutant (MT) leukemic clones have a different cell type of origin from the ancestral clone. Next, using single-cell transcriptomics in cells isolated from xenografts of two AML-iPSC lines generated from an AML patient with a subclonal KRAS G12D mutation, one capturing the RAS WT major clone and one the KRAS G12D subclone, we show that KRAS G12D leukemia stem cells (LSCs) give rise to leukemia with a more monocytic immunophenotype, while the ancestral clone within the same patient generates more leukemic cells with primitive and neutrophil markers. These results were corroborated by Genotyping of Transcriptomes (GoT) in another AML patient with subclonal NRAS G12D mutation. Recent studies have linked monocytic AML and, independently, RAS pathway mutations to poor responses to Venetoclax (VEN)-containing regiments. In view of the causal link we found between RAS mutations and monocytic disease, we evaluated the associations between monocytic AML, RAS mutations and response to VEN. We reanalyzed data from a cohort of 118 older/unfit newly diagnosed AML patients treated on a prospective clinical trial with VEN and decitabine (DEC) (NCT03404193). All outcomes were comparable between monocytic and non-monocytic groups, including overall response rate, duration of response (DOR) and overall survival. On the other hand, patients with N/KRAS mutations had significantly shorter DOR. These results support the presence of N/KRAS mutations and not the monocytic stage as predictors of inferior responses to VEN regimens. To definitely test this, we treated defined cell types generated from RAS WT and RAS MT iPSC lines - CD34+ LSC-like and CD11b+/CD68+/CD14+ monocytic cells - with VEN. In agreement with previous studies, monocytes were resistant and RAS WT LSCs were sensitive to VEN. However, strikingly RAS MT LSCs were VEN-resistant. Consistent with these, single-cell transcriptomics in patient-derived AML-iPSC cells in vivo, GoT in primary AML cells and bulk transcriptomics in synthetic iPSC-AML cells showed downregulation of BCL2 and upregulation of MCL1 in the RAS MT LSCs. This study provides the first example of a mechanistic explanation of why the timing of mutational acquisition in AML is so strongly biased. It shows for the first time that interaction between an oncogenic driver and the non-genetic developmental hematopoietic hierarchy imposes a specific LSC cell-of-origin restriction and in turn shapes the hierarchical structure of the resulting AML and critically determines therapeutic outcomes in patients. Importantly, our results have direct implications for clinical practice, as they support that RAS MT LSCs drive clinical resistance to VEN and that treatment with VEN in patients with RAS mutations can accelerate disease progression by selection of RAS MT LSCs (Figure).

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.

Plm-102, a Next Generation FLT3 Inhibitor, Shows Potent Anti-Leukemic Activity on Resistance to Gilteritinib in FLT3 Mutated Acute Myeloid Leukemia

Introduction: Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 25 ~ 30% of all acute myeloid leukemia (AML) cases, with FLT3-ITD mutations having a poor prognosis. In 2022, the FDA granted approval to Gilteritinib, a 2nd generation FLT3 inhibitor, for the treatment of relapsed or refractory AML. Although Gilteritinib has demonstrated efficacy in R/R AML patients, its response duration is limited due to the development of secondary resistance. Herein, we report a next-generation drug candidate, PLM-102, which has shown promising pre-clinical results to overcome the current unmet needs of Gilteritinib resistance, including FLT3-ITD-TKD and Ras mutations in FLT3-mutated AML. Aims: The main objective of this study is to investigate the effect of PLM-102 in in-vitro and in-vivo acquired Gilteritinib-resistant models and to elucidate the underlying signaling pathways. Methods: To establish Gilteritinib-resistant cell lines, we exposed the AML cells to FLT3 ligand at 10 ng/ml and Gilteritinib at concentrations ranging from 10 nM to 100 nM for four months. Subsequently, for the following four months, we treated the cells with Gilteritinib alone at 100 nM, omitting FLT3 ligand from the treatment. Whole Exome Sequencing (WES) and mRNA sequencing were performed on primary FLT3 mutated AML cells and Gilteritinib resistance FLT3 mutated AML cells. Subsequently, cell viability assays were carried out on both parent FLT3-mutated AML cells and Gilteritinib-resistant FLT3-mutated AML cells. To assess the activities of PLM-102 at the on-target and off-target resistant mechanisms, immunoblot studies were conducted. An in vivo study was performed using a Gilteritinib-resistant MOLM-14 AML xenograft mouse model. Results: We conducted Whole Exome Sequencing on the Gilteritinib-resistant MOLM-13 and MOLM-14 cell lines. The analysis revealed the presence of KRAS G13D and FLT3-ITD-C828G mutations in the Gilteritinib-resistant MOLM-13 cell line, and NRAS G13D and FLT3-ITD-G697R mutations in the Gilteritinib-resistant MOLM-14 cell line. In the evaluation of the efficacies of Gilteritinib and PLM-102 in Gilteritinib-resistant MOLM-14 cell lines. The GI 50 values for Gilteritinib and PLM-102 were determined to be 427 nM and 6.17 nM, respectively. In the studies with a xenograft model with Gilteritinib-resistant MOLM-14 cells (20 mg/kg, p.o.), the Tumor Growth Inhibition (TGI, %) values were determined as 71.9% vs 17.1% for PLM-102 vs Gilteritinib, respectively. These results demonstrated the superior in-vivo efficacies of PLM-102 in Gilteritinib-resistant cells. mRNA sequencing was performed on both MOLM-14 cells and Gilteritinib-resistance MOLM-14 cells, resulting in an upregulation of the cytokine-mediated signaling pathway, including chemokines, in the Gilteritinib-resistant MOLM-14 cells. Additionally, we conducted mRNA sequencing on Gilteritinib-resistant MOLM-14 cells treated with and without PLM-102 at a concentration of 10 nM. Interestingly, in the group treated with PLM-102 at 10 nM, we observed a notable decrease in the cytokine-mediated signaling pathway, including chemokines. These findings provide valuable insights into the potential mechanism by which PLM-102 exerts its anti-leukemic effect in Gilteritinib-resistant cells by modulating the cytokine-mediated signaling pathway. In Gilteritinib-resistant MOLM-14 cells harboring the NRAS G13D mutation, we confirmed hyperactivation of the MEK-ERK signaling pathway. The significantly improved effects in the combination of PLM-102 and Trametinib, a MEK inhibitor, were observed through cell viability assays and a xenograft model, further supporting their potential as a synergistic therapeutic approach. Currently, the GLP toxicological studies of PLM-102 has successfully completed, and the Investigational New Drug (IND) for human clinical studies will be submitted near future. Conclusions: Our results have confirmed that RAS-MEK-ERK signaling pathway and cytokine signaling pathway were activated by Gilteritinib resistance. Notably, PLM-102 exhibits exceptional anti-leukemic activity in Gilteritinib-resistant cell lines through the unique mechanism of inhibiting the RAS-MEK-ERK-cytokine signaling pathway. These results highlight the potential of PLM-102 as a third-generation inhibitor with favorable efficacies in patients with resistance to current FLT3 inhibitors.

Identification of Potential Inhibitors Targeting GTPase-Kirsten RAt Sarcoma Virus (K-Ras) Driven Cancers via E-Pharmacophore-Based Virtual Screening and Drug Repurposing Approach.

Mutations in the K-Ras gene are among the most frequent genetic alterations in various cancers, and inhibiting RAS signaling has shown promising results in treating solid tumors. However, finding effective drugs that can bind to the RAS protein remains challenging. This drove us to explore new compounds that could inhibit tumor growth, particularly in cancers that harbor K-Ras mutations. Our study used bioinformatic techniques such as E-pharmacophore virtual screening, molecular simulation, principal component analysis (PCA), extra precision (XP) docking, and ADMET analyses to identify potential inhibitors for K-Ras mutants G12C and G12D. In our study, we discovered that inhibitors such as afatinib, osimertinib, and hydroxychloroquine strongly inhibit the G12C mutant. Similarly, hydroxyzine, zuclopenthixol, fluphenazine, and doxapram were potent inhibitors for the G12D mutant. Notably, all six of these molecules exhibit a high binding affinity for the H95 cryptic groove present in the mutant structure. These molecules exhibited a unique affinity mechanism at the molecular level, which was further enhanced by hydrophobic interactions. Molecular simulations and PCA revealed the formation of stable complexes within switch regions I and II. This was particularly evident in three complexes: G12C-osimertinib, G12D-fluphenazine, and G12D-zuclopenthixol. Despite the dynamic nature of switches I and II in K-Ras, the interaction of inhibitors remained stable. According to QikProp results, the properties and descriptors of the selected molecules fell within an acceptable range compared to sotorasib. We have successfully identified potential inhibitors of the K-Ras protein, laying the groundwork for the development of targeted therapies for cancers driven by K-Ras mutations.

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

Deep molecular tracking over the 12-yr development of endometrial cancer from hyperplasia in a single patient.

Although the progressive histologic steps leading to endometrial cancer (EndoCA), the most common female reproductive tract malignancy, from endometrial hyperplasia are well-established, the molecular changes accompanying this malignant transformation in a single patient have never been described. We had the unique opportunity to investigate the paired histologic and molecular features associated with the 12-yr development of EndoCA in a postmenopausal female who could not undergo hysterectomy and instead underwent progesterone treatment. Using a specially designed 58-gene next-generation sequencing panel, we analyzed a total of 10 sequential biopsy samples collected over this time frame. A total of eight pathogenic/likely pathogenic mutations in seven genes, APC, ARID1A, CTNNB1, CDKN2A, KRAS, PTEN, and TP53, were identified. A PTEN nonsense mutation p.W111* was present in all samples analyzed except histologically normal endometrium. Apart from this PTEN mutation, the only other recurrent mutation was KRAS G12D, which was present in six biopsy samplings, including histologically normal tissue obtained at the patient's first visit but not detectable in the cancer. The PTEN p.W111* mutant allele fractions were lowest in benign, inactive endometrial glands (0.7%), highest in adenocarcinoma (36.9%), and, notably, were always markedly reduced following progesterone treatment. To our knowledge, this report provides the first molecular characterization of EndoCA development in a single patient. A single PTEN mutation was present throughout the 12 years of cancer development. Importantly, and with potential significance toward medical and nonsurgical management of EndoCA, progesterone treatments were consistently noted to markedly decrease PTEN mutant allele fractions to precancerous levels.

An in vitro-transcribed circular RNA targets the mitochondrial inner membrane cardiolipin to ablate EIF4G2+/PTBP1+ pan-adenocarcinoma.

In vitro-transcribed (IVT) mRNA has arisen as a rapid method for the production of nucleic acid drugs. Here, we have constructed an oncolytic IVT mRNA that utilizes human rhinovirus type 2 (HRV2) internal ribosomal entry sites (IRESs) to selectively trigger translation in cancer cells with high expression of EIF4G2 and PTBP1. The oncolytic effect was provided by a long hGSDMDc .825 T>A/c.884 A>G-F1LCT mutant mRNA sequence with mitochondrial inner membrane cardiolipin targeting toxicity that triggers mitophagy. Utilizing the permuted intron-exon (PIE) splicing circularization strategy and lipid nanoparticle (LNP) encapsulation reduced immunogenicity of the mRNA and enabled delivery to eukaryotic cells in vivo. Engineered HRV2 IRESs-GSDMDp.D275E/E295G-F1LCT circRNA-LNPs (GSDMDENG circRNA) successfully inhibited EIF4G2+/PTBP1+ pan-adenocarcinoma xenografts growth. Importantly, in a spontaneous tumor model with abnormal EIF4G2 and PTBP1 caused by KRAS G12D mutation, GSDMDENG circRNA significantly prevented the occurrence of pancreatic, lung and colon adenocarcinoma, improved the survival rate and induced persistent KRAS G12D tumor antigen-specific cytotoxic T lymphocyte responses.

Oncogenic context shapes the fitness landscape of tumor suppression

Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged—the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context—and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.

A real-world analysis of trametinib in combination with hydroxychloroquine or CDK4/6 inhibitor as third- or later-line therapy in metastatic pancreatic adenocarcinoma

BackgroundThere are no standard third-line treatment options for metastatic pancreatic ductal adenocarcinoma (mPDAC). Trametinib in combination with hydroxychloroquine (HCQ) or CDK4/6 inhibitors for pancreatic adenocarcinoma showed promising efficacy in preclinical studies. However, the regimens have not been well examined in patients with mPDAC.MethodsPatients with mPDAC who received the combination of trametinib and HCQ or CDK4/6 inhibitors as third- or later-line therapy were reviewed. The efficacy and prognosis were further analyzed.ResultsA total of 13 mPDAC patients were enrolled, of whom 8 and 5 patients were treated with trametinib plus HCQ or a CDK4/6 inhibitor (palbociclib or abemaciclib), respectively. All enrolled patients had either KRAS G12D or G12V mutations and had received a median of 3 prior lines of therapy (range, 2–6). The median trametinib treatment duration was 1.4 months. Of the 10 patients with measurable disease, only 1 patient achieved stable disease, and the remaining patients had progressive disease. Moreover, in patients treated with trametinib plus HCQ and a CDK4/6 inhibitor, the median progression-free survival was 2.0 and 2.8 months, respectively, and the median overall survival was 4.2 and 4.7 months, respectively. Moreover, 5 (50%) patients experienced grade 3–4 adverse events in 10 patients with available safety data.ConclusionsThe combination of trametinib and HCQ or CDK4/6 inhibitors may not be an effective later-line treatment for mPDAC, and the current preliminary findings need to be confirmed by other studies with larger sample sizes.

LBA33 A first-in-human phase I study of a novel KRAS G12D inhibitor HRS-4642 in patients with advanced solid tumors harboring KRAS G12D mutation

LBA33 A first-in-human phase I study of a novel KRAS G12D inhibitor HRS-4642 in patients with advanced solid tumors harboring KRAS G12D mutation

Identification of neoepitope reactive T-cell receptors guided by HLA-A*03:01 and HLA-A*11:01 immunopeptidomics

BackgroundTumor-specific mutated proteins can create immunogenic non-self, mutation-containing ‘neoepitopes’ that are attractive targets for adoptive T-cell therapies. To avoid the complexity of defining patient-specific, private neoepitopes, there has been major...

The Effect of Chemotherapeutic Agents on Survival in Metastatic Non-Small-Cell Lung Cancer with KRAS Mutation

Abstract Introduction KRAS mutation is observed in up to 30% of non-small-cell lung cancer (NSCLC) cases and is corelated with a poor prognosis. In the cases with KRAS p.G12C mutation and first-line chemotherapy (± immunotherapy) resistance, a targeted drug option is available. Objectives Our study aimed to examine the correlation between first-line chemotherapy agents and treatment response in patients with KRAS-mutated metastatic NSCLC. Materials and Methods Retrospective database searches were performed on cases diagnosed with metastatic NSCLC at our center between January 2019 and December 2021 that were found to be KRAS mutation positive using the next-generation sequencing (NGS) approach. The cases were classified into five subgroups based on the chemotherapy regimens (platinum + gemcitabine, platinum + taxane, platinum + pemetrexed, platinum + vinorelbine, and others). The clinical and demographic data of 41 cases were analyzed retrospectively, and survival analyses were performed using the Kaplan–Meier method. Results Thirty-seven of 41 patients (90.2%) were males, and 27 (65.9%) had adenocarcinoma histology. The most prevalent mutation was KRAS G12C, with 12 cases (29.2%), followed by KRAS G12V, with 9 cases (21.9%). Other mutations were as follows: KRAS G12D 4 (9%), KRAS G13C 3 (7.3%), KRAS G12A 2 (4.8%), KRAS G12R 2 (4.8%), KRAS Q61H 2 (4.8%), KRAS Q61L 2 (4.8%), KRAS V14I 2 (4.8%), KRAS A146T 1 (2.4%), KRAS G13G 1 (2.4%), and KRAS G1C 1 (2.4%). The median progression-free survival (mPFS) for all groups was 4.6 months (95% confidence interval [CI]: 2.7-6.5), and there were no statistically significant differences between the groups (p = 0.121). The median overall survival (mOS) for all groups was 9.3 months (95% CI: 3.8–14.5), and there were no statistically significant differences between the groups (p = 0.805). Conclusions OS and PFS analyses showed no differences between platinum + taxane, platin + pemetrexed, platinum + gemcitabine, and platin + vinorelbine used in first-line treatments for KRAS mutant NSCLC cases. We believe that patient-specific characteristics may be a determining factor in selecting chemotherapy for this patient population.

Molecular Pattern and Clinical Implications of KRAS/NRAS and BRAF Mutations in Colorectal Cancer.

The aim of our study was to evaluate the incidence of KRAS/NRAS and BRAF mutations, analyze molecular patterns, and investigate associations with clinical parameters of these mutations in CRC KRAS/NRAS and BRAF mutations analyzed by next-generation sequencing. The detection rates of these mutations and patients' demographics were recorded and the relationship between them was evaluated using the chi-square test. KRAS mutation was detected in 332 of 694 patients, while the mutation rates in KRAS exons 2/3 and 4 were 39.6%/3.2% and 5%, respectively. The most common mutation pattern was KRAS G12D. Five atypical variants were detected: V14I in KRAS exon 2, A18D, Q22K and T50I in exon 3, and T148P in exon 4. NRAS mutation was detected in 29 (4.5%) patients. One atypical variant L80W was detected in NRAS exon 3. BRAF mutation was seen in 37 (5.3%) patients, with BRAFV600E (83.8%) being the most common mutation pattern. NRAS mutation was significantly more frequent in patients > 64 years of age, BRAF mutation in women, and NRAS/BRAF mutations in right colon tumors. Grouping BRAF mutations into BRAFV600E and BRAFnon-V600E and their analysis according to specific tumor localizations showed that all four BRAFnon-V600E mutations originated in the rectum. In our study, KRAS exon 2 and other RAS mutation rates were higher than in the literature, while the BRAF v.600E mutation rate was similar. NRAS and BRAF mutations were significantly more frequent in the right colon. BRAF mutation was more common in women and in the right colon.

Structure-Based Design and Synthesis of Potent and Selective KRAS G12D Inhibitors.

KRAS G12D mutation has been found in approximately 45% of pancreatic ductal adenocarcinoma (PDAC) cases, making it an attractive therapeutic target. Through structure-based drug design, a series of potent and selective KRAS G12D inhibitors were designed. The lead compound, ERAS-5024, inhibited ERK1/2 phosphorylation and cell proliferation in three-dimensional Cell-Titer Glo assays in AsPC-1 PDAC cells with single-digit nanomolar potency and caused tumor regression in the in vivo efficacy studies. We describe here the details of the design and synthesis program that led to the discovery of ERAS-5024.

Salvianolic acid F suppresses KRAS-dependent lung cancer cell growth through the PI3K/AKT signaling pathway

BackgroundKRAS mutation is a common driver of NSCLC, and there is a high proportion of lung cancer patients with KRAS G12C and G12D mutation. KRAS was previously considered an "undruggable" target, but the first KRAS G12C mutation-targeted drug AMG510, entered the market in 2021. However, treatments for G12D mutant tumors remain to be discovered. Salvianolic acid F (SalF), a monomer derived from the traditional Chinese medicine Salvia miltiorrhiza (SM), and KRAS had high binding affinity, especially for KRAS G12D. There is an urgent need to investigate effective and safe novel targeted therapies against KRAS G12D-driven NSCLC. MethodsTo evaluate the anticancer effect of SalF, we used KRAS-overexpressing lung cancer cells in vitro, a subcutaneous transplant tumor model, and KRAS G12D mice model in vivo. Then, the binding effect of SalF and KRAS was investigated using molecular docking, proteolytic assays and protein thermal shift assays. More critically, the PI3K/AKT signaling pathway in the lung was investigated utilizing RT-qPCR and Western Blotting. ResultsThis is the first study to evaluate the anticancer effect of SalF on KRAS-overexpressing lung cancer cells or KRAS G12D lung tumors in vivo. We demonstrated that SalF inhibits OE-KRAS A549 cell migration, proliferation and promotes apoptosis in vitro. In addition, we used a subcutaneous transplant tumor model to show that SalF suppresses the growth of lung cancer cells in vivo. Interestingly, our group found that SalF was strongly bound to G12D and could decrease the stability and promoted the degradation of the KRAS G12D mutant through molecular docking, proteolytic assays and protein thermal shift assays. Further research demonstrated that in the KrasG12D mice model, after SalF treatment, the number and size of mouse lung tumors were significantly reduced. More importantly, SalF can promote apoptosis by inhibiting downstream PI3K/AKT signaling pathway activation. ConclusionSalF activated apoptosis signaling pathways, suppressed anti-apoptotic genes, and inhibited lung cancer cell growth. These datas suggested that SalF could effectively inhibit the growth of lung tumors with KRAS G12D mutation. SalF may be a novel inhibitor against KRAS G12D, providing a strong theoretical basis for the clinical treatment of lung cancer with KRAS mutations.