Common Oncogenic Mutations Research Articles

Selective targeting of oncogenic hotspot mutations of the HER2 extracellular domain.

Oncogenic mutations in the extracellular domain (ECD) of cell-surface receptors could serve as tumor-specific antigens that are accessible to antibody therapeutics. Such mutations have been identified in receptor tyrosine kinases including HER2. However, it is challenging to selectively target a point mutant, while sparing the wild-type protein. Here we developed antibodies selective to HER2 S310F and S310Y, the two most common oncogenic mutations in the HER2 ECD, via combinatorial library screening and structure-guided design. Cryogenic-electron microscopy structures of the HER2 S310F homodimer and an antibody bound to HER2 S310F revealed that these antibodies recognize the mutations in a manner that mimics the dimerization arm of HER2 and thus inhibit HER2 dimerization. These antibodies as T cell engagers selectively killed a HER2 S310F-driven cancer cell line in vitro, and in vivo as a xenograft. These results validate HER2 ECD mutations as actionable therapeutic targets and offer promising candidates toward clinical development.

Treatment of a mutant KRAS lung cancer cell line with polyisoprenylated cysteinyl amide inhibitors activates the MAPK pathway, inhibits cell migration and induces apoptosis.

KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated Cysteinyl amide Inhibitors (PCAIs) constitute a group of potential cancer therapy agents that we designed to specifically disrupt and suppress hyperactive G-protein signaling, such as that caused by mutated RAS proteins. Here we determine the effects of PCAIs on the viability, G-protein levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutated, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 μM, respectively. Western blotting was used to determine the effect of the PCAIs on the phosphorylation levels of MAPK pathway enzymes. After 48 h exposure to 5 μM of the PCAIs, NSL-YHJ-2-46, the MAPK proteins BRAF, MEK1/2, ERK1/2, and p90RSK were activated through phosphorylation by 90, 190, 150 and 120%, respectively. However, CRAF/RAF1 phosphorylation decreased by 40%, suggesting significant changes in the KRAS/MAPK signaling patterns. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed inhibition of cell migration and invasion, and spheroid invasion observed on exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46. Treatment with 5 μM of NSL-YHJ-2-27 suppressed full-length inactive caspase 3 and 7 levels by 72 and 91%, respectively. An analysis of cells treated with the fluorescently labeled active caspase 3/7 irreversible inhibitor, CaspaTagTM Caspase-3/7 in situ reagent revealed a 124% increase in active caspase at 3 μM over controls. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway. Given the profound increases observed in RPS6KA1/p90RSK phosphorylation, future work will involve a determination whether the proapoptotic isoforms of RPS6KA1/p90RSK are phosphorylated due to the PCAIs treatments. These results support the potential use of the PCAIs as targeted therapies against cancers with KRAS mutations.

Pathways and mechanism of MRTX1133 binding to KRAS G12D elucidated by molecular dynamics simulations and Markov state models

KRAS G12D is the most common oncogenic mutation identified in several types of cancer. Therefore, design of inhibitors targeting KRAS G12D represents a promising strategy for anticancer therapy. MRTX1133 is a highly potent inhibitor (approximate experiment Kd ≈ 0.0002 nM) of KRAS G12D and is currently in Phase 1/2 study, however, pathways of the compound binding to KRAS G12D has remained unknown, and the mechanism underlying the complicated dynamic process are challenging to capture experimentally, which hinder the structure-based anti-cancer drug design. Here, using MRTX1133 as a probe, unbiased molecular dynamics (MD) was used to simulate the process of MRTX1133 spontaneously binding to KRAS G12D. In six of 42 independent MD simulation (a total of 99 μs), MRTX1133 was observed to successfully associate with KRAS G12D. The kinetically metastable states refer to the potential pathways of MRTX1133 binding to KRAS G12D were revealed by Markov state models (MSM) analysis. Additionally, 8 key residues that are essential for MRTX1133 recognition and tight binding at the preferred low energy states were identified by MM/GBSA analysis. In sum, this study provides a new perspective on understanding the pathways and mechanism of MRTX1133 binding to KRAS G12D.

Resolving Discrepancies in Idylla BRAF Mutational Assay Results Using Targeted Next-Generation Sequencing.

BRAF mutation identification is important for the diagnosis and treatment of several tumor types, both solid and hematologic. Rapid identification of BRAF mutations is required to determine eligibility for targeted BRAF inhibitor therapy. The Idylla BRAF mutation assay is a rapid, multiplex allele-specific PCR test designed to detect the most common oncogenic BRAF V600 mutations in formalin-fixed paraffin-embedded (FFPE) tissue samples. Here, we describe the validation of the Idylla BRAF mutation assay in our laboratory. During routine clinical practice, we noticed cases in which BRAF V600 mutations were identified with unusual amplification curves, with three cases displaying a delayed amplification within a double amplification pattern and two false-positive calls. We therefore initiated a quality improvement effort to systematically and retrospectively evaluate next-generation sequencing (NGS)-tested cases with BRAF mutations identified within five amino acids of BRAF codon V600 and did not identify additional false-positive cases. We hypothesize that late amplification in a double amplification pattern may represent non-specific amplification, whereas cases displaying single delayed amplification curves may stem from the presence of either non-V600 variants, very low-level V600 variants, cytosine deamination artifacts, and/or non-specific amplification by an allele-specific PCR primer. Regardless, we recommend that Idylla BRAF cases with non-classical amplification curves undergo reflex NGS testing. These findings are likely relevant for other Idylla assays interrogating hotspot mutations in genes such as EGFR, IDH1/2, KRAS, and NRAS.

Abstract LB044: Selective therapeutic antibodies against oncogenic mutations of the HER2 extracellular region

Abstract Oncogenic mutations of HER2 have been identified in HER2-low tumors. Such mutations in the extracellular region could serve as tumor-specific antigens that are accessible to antibody therapeutics. Although multiple antibody therapeutics have been successfully developed against cancers that overexpress wild-type HER2, none is specifically applicable to treating tumors driven by HER2 mutants expressed at a low level. Here, we developed antibodies exquisitely selective to HER2 S310F and S310Y, two most common oncogenic mutations in the HER2 extracellular region. Cryo-EM structures of the homodimer of HER2 S310F and an antibody bound to HER2 S310F revealed that these antibodies recognize the mutations in a manner that mimic the dimerization arm of HER and inhibit HER2 dimerization. These antibodies as T-cell engagers selectively killed a HER2 S310F-driven cancer cell line in vitro and in a mouse xenograft model. These results validate HER2 extracellular region mutations as actionable therapeutic targets and offer promising candidates toward clinical development. More generally, they suggest that point mutations in the extracellular region of cell-surface receptors as actionable targets for anti-cancer therapy. Citation Format: Injin Bang, Takamitsu Hattori, Nadia Leloup, Alexis Corrado, Atekana Nyamaa, Akiko Koide, Ken Geles, Elizabeth Buck, Shohei Koide. Selective therapeutic antibodies against oncogenic mutations of the HER2 extracellular region [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 LB044.

Abstract 3591: Upregulation of CXCL8 is an early step in the oncogenic transition of human thyroid cells by BRAFV600E

Abstract Introduction: BRAFV600E is one of the most common oncogenic mutations in human thyroid cancers, especially in papillary thyroid carcinoma (PTC). However, the early molecular changes caused by this mutation have not been studied previously in human thyrocytes in vitro. Methods: Primary cultures of cells derived from noncancerous thyroid tissue of patients (at least n=3) were infected with BRAFV600E adenovirus to mimic the mutated condition. Changes induced by the infection were measured at early times (<24h). Transcriptome analysis was performed on infected thyrocytes (n=6) to determine the highest differentially expressed genes between the control and infected groups, which were then verified at the gene and protein levels. RNAi downregulation experiments were performed to determine the cross-regulatory function of the genes. Results: The induction of BRAFV600E mutation, confirmed at the protein level, increased proliferation of the cells, and morphological changes were observed at approximately 24h post-infection. The decreased expression of thyroid-specific genes, viz. TSHR, TG, and TPO were observed after >72h post-introduction of BRAFV600E mutation. Transcriptome and protein expression analysis of BRAFV600E-positive thyrocytes showed the overexpression of chemokines CXCL8 and CXCL12, and upregulation of endothelial cell-specific molecule 1 (ESM1). These molecules are known to be associated with increased neoangiogenesis and proliferation. Functional studies showed that siRNA knockdown of CXCL8 decreased the expressions of CXCL12 and ESM1, whereas knockdown of either CXCL12 or ESM1 did not decrease the expression of CXCL8. Conclusion: Increased proliferation, de-differentiation, upregulation of chemokines, and endothelial-specific factor are effects associated with BRAFV600E-driven thyroid carcinogenesis. CXCL8 appears to play an important early regulatory role in this process. Citation Format: Rhitajit Sarkar, Priyanka Bolel, Joanna Klubo-Gwiezdzinska, Marvin C. Gershengorn. Upregulation of CXCL8 is an early step in the oncogenic transition of human thyroid cells by BRAFV600E [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 3591.

Abstract 893: Studying the impact of CNVs on expression at single-cell resolution in HGSOC using autoencoders

Abstract High-grade serous ovarian cancer (HGSOC) is one of the most lethal gynecological malignancies. Lack of common targetable oncogenic mutations has complicated the development of directed therapies to combat emerging resistance. This malignancy is mainly characterized by the mutation of gene TP53, which promotes genome instability for the emergence of extensive copy number variations (CNVs). However its impact on gene expression at the single-cell level is not well understood. In this study, we aim to investigate the effect of CNVs on transcriptomic signatures by taking advantage of variational autoencoders (VAE) ability for dimensionality reduction, unsupervised learning and feature extraction. The use of VAEs is becoming more popular for the analysis of scRNAseq data, and scVI is one of the most versatile VAE applications performing wide variety of tasks. Here, we used single-cell RNA sequencing (scRNA-seq) data from 90 longitudinal samples of 64 HGSOC patients and inferred CNVs in each cell using inferCNV, an established computational pipeline. Then, we modified scVI algorithm to allow the VAE to reconstruct CNVs from a latent space originated from gene expression profiles and viceversa. Our models were capable of reconstructing CNV profiles accurately from expression data and also remove batch effect. From these models we could observe how, after the integration of genomic information, the latent clusters produced from the transcriptomic space were influenced by the amplifications or deletions of certain genomic regions. Moreover, some of these clusters were characterized by the alteration of important oncogenes in HGSOC, such as KRAS and CCNE1, and allowed us to focus on the transcriptomic consequences of their amplifications. With the results from the approach presented here, we gained a more comprehensive picture of the impact of genomic alterations on HGSOC. As future work, we plan to validate of this results on external cohorts and link the identified signatures to clinically relevant features such as prognosis or chemotherapy response. Citation Format: Matias Marin Falco, Teemu Närhi, Erdogan Pekcan Erkan, Johanna Hynninen, Anna Vähärautio. Studying the impact of CNVs on expression at single-cell resolution in HGSOC using autoencoders [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 893.

Abstract B003: Inhibition of cell growth and migration by polyisoprenylated cysteinyl amide inhibitors of a mutant KRAS Black American lung cancer cell line involves MAPK pathway activation and disruption of the cytoskeleton

Abstract KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated Cysteinyl Amide Inhibitors (PCAIs) constitute a group of potential cancer therapy agents that we designed to specifically disrupt and suppress hyperactive G-protein signaling, as caused by mutated RAS proteins. Here we investigate the effects of PCAIs on the viability, G-protein levels, downstream mediators, cytoskeletal structure, focal adhesion protein and apoptosis-related proteins on the KRAS-mutated, Black American-derived lung adenocarcinoma cell line, NCI-H23. Western blotting was used to determine the effect of the PCAIs on the phosphorylation levels of MAPK pathway enzymes. After 48-hour exposure to 5 μM of the PCAIs, 57 ± 3.6 to 150 ± 4.1 % increases of BRAF, MEK1/2, ERK1/2, and p90RSK phosphorylation along with a 60 ± 6.5 to 78 ± 4.0 % decrease of pRAF1 phosphorylation were observed, indicating significant changes in RAS signaling. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 ± 5.5 and 76 ± 2.0 %, respectively. The depletion of these key cytoskeletal proteins, and collapse of F-actin and vinculin punctates, may account for the observed inhibition of cell migration after 72-hour exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46, respectively. The invasion area of 3D NCI-H23 spheroids after treatment with 10 μM of NSL-YHJ-2-27 and NSL-YHJ-2-46 decreased by 87 ± 0.1% and 92 ± 0.3 %, respectively. Treatment with 0.5 μM NSL-YHJ-2-27 and NSL-YHJ-2-46 caused cell rounding and reduction of mean cell areas by 30 ± 5.0 and 42 ± 0.6 %, respectively. Mean cell area occupied by vinculin punctates decreased by 51 ± 2.6 % and 31 ± 2.8 % after exposure to 0.5 μM NSL-YHJ-2-27 and NSL-YHJ-2-46, respectively. NSL-YHJ-2-27 (5 μM) depleted full-length inactive caspase 3 and 7 levels by 72 ± 5.8 and 91 ± 7.0 %, respectively. These findings show the direct effects of the PCAIs on the KRAS signaling pathway thereby supporting their continuous development as potential targeted therapies for cancers with RAS mutations. Citation Format: Kweku Ofosu-Asante. Inhibition of cell growth and migration by polyisoprenylated cysteinyl amide inhibitors of a mutant KRAS Black American lung cancer cell line involves MAPK pathway activation and disruption of the cytoskeleton [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B003.

Abstract A038: Selective therapeutic antibodies against oncogenic mutations of HER2 ectodomain

Abstract Dysregulation of HER2 is a well-established mechanism of oncogenesis. Small molecule drugs targeting its kinase domain, and antibody-based drugs such as trastuzumab and pertuzumab targeting its ectodomain have shown efficacy against HER2-overexpression tumors. Separately from HER2 overexpression, oncogenic mutations in the ectodomain in HER2 have been identified in several tumors. As disease drivers that are readily accessible to biologics, these mutants are potentially attractive but still unvalidated therapeutic targets. Current anti-HER2 therapeutic antibodies are indiscriminatory of the mutational state of HER2 and their cross-reactivity to wild-type HER2 can cause toxicity. Here, we have developed antibodies exquisitely selective to HER2 S310F and S310Y, two most common oncogenic mutations in the HER2 ectodomain. These antibodies bound to both HER2 S310F/Y with >100-fold selectivity over the wild-type. Cryo-EM structures of the homodimer of HER2 S310F and an antibody bound to HER2 S310F revealed that these antibodies mimic the dimerization arm of HER2 and inhibit dimerization. The developed antibodies as T cell engagers selectively killed a HER2 S310F-driven cancer cell line in vitro. These results establish the feasibility of targeted therapy against S310F/Y-driven cancers and offer promising candidates toward clinical development. Citation Format: Injin Bang, Takamitsu Hattori, Nadia Leloup, Alexis Corrado, Atekana Nyamaa, Akiko Koide, Ken Geles, Elizabeth Buck, Shohei Koide. Selective therapeutic antibodies against oncogenic mutations of HER2 ectodomain [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 A038.

Abstract B137: Selective inhibition of the active state of KRASG12V with the non-covalent, tri-complex inhibitor RM-048

Abstract The KRASG12V mutation is the second most common oncogenic RAS mutation and is frequently observed in pancreatic, lung, and colorectal cancers. Developing a selective inhibitor of KRASG12V presents significant drug discovery challenges. The intrinsic GTP hydrolysis rate of KRASG12V is about 12-fold lower than that of KRASG12C, further biasing the cellular KRASG12V pool to the active, GTP-bound (“RAS(ON)”) state and emphasizing the importance of targeting the KRASG12V(ON) state for maximal suppression of this oncogenic driver. Additionally, achieving selectivity over wild-type RAS with small molecules is difficult because the valine residue introduced by KRASG12V is neither amenable to covalent inhibition nor to formation of polar, non-covalent interactions. We have previously applied our tri-complex inhibitor platform, which uses chemical re-remodeling of the cellular chaperone cyclophilin A (CypA) in order to bind to undruggable surfaces, to design mutant-selective inhibitors targeting KRASG12C, KRASG13C, KRASG12D, and KRASQ61H, in addition to the RASMULTI inhibitor RMC-6236. Here, we describe RM-048, a potent, selective, and oral tri-complex inhibitor of KRASG12V(ON). RM-048 binds to CypA with high affinity to form a binary complex. Binding to CypA creates a neomorphic interface that forms a selective, high-affinity, non-covalent interaction with KRASG12V(ON). The resulting tri-complex sterically blocks effector binding to KRASG12V(ON), thereby inhibiting downstream signaling. RM-048 potently suppressed ERK phosphorylation and proliferation in KRASG12V mutant cancer cells. In preclinical species, RM-048 showed good bioavailability, dose-proportional exposure, and low clearance, allowing for oral dosing. In human xenograft tumors harboring KRASG12V mutations, a single dose of RM-048 induced dose-dependent, deep, and durable suppression of RAS pathway signaling in vivo. Repeated daily oral administration of RM-048 was well tolerated and demonstrated profound anti-tumor activity, driving tumor regressions across a panel of KRASG12V xenograft models, including NSCLC, PDAC, and CRC models. RM-048 complements the mutant-selective tri-complex KRAS(ON) inhibitors RMC-6291 (KRASG12C) and RMC-9805 (KRASG12D) by potentially providing a mutant-selective inhibitor option for patients with tumors harboring one of the most frequent RAS mutations. Preclinical combination studies with RAS companion inhibitors, including the RASMULTI inhibitor RMC-6236, are ongoing. Citation Format: Bianca J Lee, Jim Cregg, Anne Edwards, Jerry Chen, Andre Eriksson, Emily Tonogai, Aidan Tomlinson, Kyle Seamon, Mariela Moreno Ayala, Nataliya Tovbis Shifrin, Nilufar Montazer, Kate Sanders, Jun Huang, Kang-Jye Chou, Benjamin Madej, Yang Liu, Zhican Wang, Zhengping Wang, John Knox, Elena Koltun, Jingjing Jiang, Adrian Gill, Jacqueline A.M. Smith. Selective inhibition of the active state of KRASG12V with the non-covalent, tri-complex inhibitor RM-048 [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 B137.

LGN loss randomizes spindle orientation and accelerates tumorigenesis in PTEN-deficient epidermis.

Loss of cell polarity and disruption of tissue organization are key features of tumorigenesis that are intrinsically linked to spindle orientation. Epithelial tumors are often characterized by spindle orientation defects, but how these defects impact tumor formation driven by common oncogenic mutations is not fully understood. Here, we examine the role of spindle orientation in adult epidermis by deleting a key spindle regulator, LGN, in normal tissue and in a PTEN-deficient mouse model. We report that LGN deficiency in PTEN mutant epidermis leads to a threefold increase in the likelihood of developing tumors on the snout, and an over 10-fold increase in tumor burden. In this tissue, loss of LGN alone increases perpendicular and oblique divisions of epidermal basal cells, at the expense of a planar orientation of division. PTEN loss alone does not significantly affect spindle orientation in these cells, but the combined loss of PTEN and LGN fully randomizes basal spindle orientation. A subset of LGN- and PTEN-deficient animals have increased amounts of proliferative spinous cells, which may be associated with tumorigenesis. These results indicate that loss of LGN impacts spindle orientation and accelerates epidermal tumorigenesis in a PTEN-deficient mouse model.

Targeting lung cancer with clinically relevant EGFR mutations using anti-EGFR RNA aptamer

A significant fraction of non-small cell lung cancer (NSCLC) cases are due to oncogenic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). Anti-EGFR antibodies have shown limited clinical benefit for NSCLC, whereas tyrosine kinase inhibitors (TKIs) are effective, but resistance ultimately occurs. The current landscape suggests that alternative ligands that target wild-type and mutant EGFRs are desirable for targeted therapy or drug delivery development. Here we evaluate NSCLC targeting using an anti-EGFR aptamer (MinE07). We demonstrate that interaction sites of MinE07 overlap with clinically relevant antibodies targeting extracellular domain III and that MinE07 retains binding to EGFR harboring the most common oncogenic and resistance mutations. When MinE07 was linked to an anti-c-Met aptamer, the EGFR/c-Met bispecific aptamer (bsApt) showed superior labeling of NSCLC cells invitro relative to monospecific aptamers. However, dual targeting invivo did not improve the recognition of NSCLC xenografts compared to MinE07. Interestingly, biodistribution of Cy7-labeled bsApt differed significantly from Alexa Fluor 750-labeled bsApt. Overall, our findings demonstrate that aptamer formulations containing MinE07 can target ectopic lung cancer without additional stabilization or PEGylation and highlights the potential of MinE07 as a targeting reagent for the recognition of NSCLC harboring clinically relevant EGFRs.

Targeting oncogenic KRAS in non-small cell lung cancer with EGFR aptamer-conjugated multifunctional RNA nanoparticles

KRAS mutations are one of the most common oncogenic driver mutations in human cancers, including non-small cell lung cancer (NSCLC), and have established roles in cancer pathogenesis and therapeutic resistance. The development of effective inhibitors of mutant KRAS represents a significant challenge. Three-way junction (3WJ)-based multi-functional RNA nanoparticles have the potential to serve as an effective invivo siRNA delivery platform with the ability to enhance tumor targeting specificity and visualize biodistribution through an imaging moiety. Herein, we assembled novel EGFRapt-3WJ-siKRASG12C mutation targeted nanoparticles to target EGFR-expressing human NSCLC harboring a KRASG12C mutation to silence KRASG12C expression in a tumor cell-specific fashion. We found that EGFRapt-3WJ-siKRASG12Cnanoparticles potently depleted cellular KRASG12C expression, resulting in attenuation of downstream MAPK pathway signaling, cell proliferation, migration/invasion ability, and sensitized NSCLC cells to chemoradiotherapy. Invivo, these nanoparticles induced tumor growth inhibition in KRASG12C NSCLC tumor xenografts. Together, this study suggests that the 3WJ pRNA-based platform has the potential to suppress mutant KRAS activity for the treatment of KRAS-driven human cancers, and warrants further development for clinical translation.

The oncogenic function of PLAGL2 is mediated via ASCL2 and IGF2 and a Wnt-independent mechanism in colorectal cancer.

Colorectal cancer (CRC) tumorigenesis and progression are linked to common oncogenic mutations, especially in the tumor suppressor APC, whose loss triggers the deregulation of TCF4/β-Catenin activity. CRC tumorigenesis is also driven by multiple epimutational modifiers such as transcriptional regulators. We describe the common (and near-universal) activation of the zinc finger transcription factor and Let-7 target PLAGL2 in CRC and find that it is a key driver of intestinal epithelial transformation. PLAGL2 drives proliferation, cell cycle progression, and anchorage-independent growth in CRC cell lines and nontransformed intestinal cells. Investigating effects of PLAGL2 on downstream pathways revealed very modest effects on canonical Wnt signaling. Alternatively, we find pronounced effects on the direct PLAGL2 target genes IGF2, a fetal growth factor, and ASCL2, an intestinal stem cell-specific bHLH transcription factor. Inactivation of PLAGL2 in CRC cell lines has pronounced effects on ASCL2 reporter activity. Furthermore, ASCL2 expression can partially rescue deficits of proliferation and cell cycle progression caused by depletion of PLAGL2 in CRC cell lines. Thus, the oncogenic effects of PLAGL2 appear to be mediated via core stem cell and onco-fetal pathways, with minimal effects on downstream Wnt signaling.NEW & NOTEWORTHY A Let-7 target called PLAGL2 drives oncogenic transformation via Wnt-independent pathways. This work illustrates the robust effects of this zinc finger transcription factor in colorectal cancer (CRC) cell lines and nontransformed intestinal epithelium, with effects mediated, in part, via the direct target genes ASCL2 and IGF2. This has implications for the role of PLAGL2 in activation of onco-fetal and onco-stem cell pathways, contributing to immature and highly proliferative phenotypes in CRC.

AFNT-211: An FAS-41BB–enhanced TCR-T cell therapy with stem-like properties targeting KRAS G12V-expressing solid tumors.

2543 Background: KRAS is the most common oncogenic driver mutation in solid tumors, promoting the initiation and progression of many uncurable cancers, including colorectal, pancreatic and lung cancer. While small molecule inhibitors to KRASG12C mutations have been approved, there are no targeted therapies available for patients with highly prevalent KRASG12V mutations. TCR-T cell therapies have demonstrated remarkable responses in clinical trials, but their durability has been limited by the immunosuppressive tumor microenvironment (TME). AFNT-211 is an autologous T cell therapy engineered to express an HLA-A*11:01 KRASG12V-specific TCR, further enhanced with CD8α/β coreceptor and a FAS-41BB switch receptor to drive T cell persistence and durable clinical responses. CD8α/β coreceptor enables a coordinated CD4+/CD8+ T cell response and FAS-41BB converts the FAS ligand (FASL) TME death signal into a costimulatory signal through 41BB activation. Methods: AFNT-211 was assessed for efficacy in vitro against a panel of KRASG12V-expressing tumor cell lines and in vivo using human xenograft mouse models. In vitro safety studies were performed to assess potential cross-reactivity, alloreactivity, and cytokine-independent growth. The clinical manufacturing process consists of autologous CD4+/CD8+ T cells transduced with lentivirus and expanded using culture conditions that drive robust expansion while preserving stem-like properties. Phenotypic and functional analyses of AFNT-211 were performed using flow cytometry and cell-based assays. Results: Coculture of AFNT-211 with a panel of KRASG12V-expressing tumor cell lines led to significant effector cytokine secretion, T cell proliferation, and tumor cell killing. The CD8α/β coreceptor enabled CD4+ T cell recognition of KRASG12V and greater overall cytotoxicity. The FAS-41BB switch receptor dramatically augmented the magnitude and durability of the anti-tumor response against FASL-expressing tumor cells. XScan mutagenesis and potential off-target peptide testing revealed no significant cross-reactivities. No alloreactivity was observed against a panel of lymphoblastoid cell lines presenting the most frequent HLA types in the US population. Potent anti-tumor response was observed in vivo in a mouse xenograft model. Affini-T’s Thrive manufacturing platform used for the production of AFNT-211 consistently delivers >30-40e9 TCR-engineered T cells with a high frequency of naïve and central memory T cells expressing negligible markers of exhaustion. Conclusions: The AFNT-211 manufacturing process generates a large number of TCR-T cells with desirable stem-like properties optimized for clinical dosing. Preclinical data demonstrated a potent and safe profile for AFNT-211 that supports clinical development in HLA-A*11:01 patients with advanced/metastatic solid tumors harboring a KRAS G12V mutation.

Abstract IA11: Targeting IDH-mutant myeloid leukemia by disabling metabolic dependencies

Abstract Somatic mutations in catalytic arginine residues of IDH1 and IDH2 contribute to the pathogenesis of acute myeloid leukemia (AML) through the production of the oncometabolite (R)-2-hydroxyglutarate (R-2-HG). Accumulating R-2HG causes epigenetic alterations by impairing αKG-dependent dioxygenases. Allosteric inhibitors of mutant IDH suppress R-2HG production and induce differentiation of leukemic blasts. These findings provide the basis of two targeted therapies for AML (ivosidenib and enasidenib), establishing the first example of personalized therapy based on cancer metabolism. However, the low response rate and the development of resistance to IDHmut inhibitors are major clinical challenges. Moreover, therapy resistance to IDH inhibition can occur through multiple mechanisms, including the selection of R-2HG-restoring second-site mutations or other sub-clonal mutations. These findings underscore the importance of developing more effective approaches for the treatment of IDH-mutant MDS and AML patients. Here we developed a panel of isogenic myeloid leukemia cell lines containing the most common IDH1 or IDH2 oncogenic mutations by CRISPR base editing. By saturation mutagenesis of IDH single-amino acid variants, we identified a repertoire of IDH second-site mutations responsible for therapy resistance through disabling uncompetitive enzyme inhibition. Recurrent mutations at NADPH binding sites within IDH heterodimers act in cis or trans to prevent the formation of stable enzyme-inhibitor complexes, restore R-2HG production in the presence of inhibitors, and drive therapy resistance in IDH-mutant AML cells and patients. Moreover, by transcriptomic and epigenomic profiling of base-edited isogenic cells, we identified a new regulatory axis involving the CD44-mediated rewiring of intracellular metabolism, which is required for mutant IDH-catalyzed R-2-HG production. More importantly, primary and resistant IDH-mutant leukemia cells are sensitive to combined IDH inhibition and genetic or pharmacologic inactivation of CD44 in vitro and in vivo. Taken together, our findings uncover a cell adhesion molecule-mediated metabolic signaling pathway as a new targetable dependency of IDH-mutant leukemia cells, with therapeutic implications for more effective treatment of primary and resistant IDH-mutant myeloid malignancies. Citation Format: Jian Xu. Targeting IDH-mutant myeloid leukemia by disabling metabolic dependencies [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr IA11.

Abstract A036: Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation

Abstract The RAS-RAF-MEK-ERK signaling axis is upregulated in many human cancers due to its central role in cell growth, differentiation and survival. Oncogenic transformation frequently arises from RAS, BRAF and MEK mutations that activate the pathway. In the clinic, similar transformations also recur as resistance mutations to KRAS.G12C inhibitor treatment. BRAF mutations at V600 (Class I) activate BRAF by releasing the kinase’s activation segment and rendering BRAF activity dimerization-independent. Yet, it remains unclear how a diverse set of dimerization-dependent BRAF mutations (Class II/III) activate the pathway. MEK mutations are assumed to bypass the RAF node entirely. We previously reported a 2.9-Å-resolution crystal structure of human BRAF kinase domain (BRAFKD) in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that stabilize an inactive, pre-signaling monomeric conformation of BRAFKD and explain how ATP breaks RAF dimers in solution. Surprisingly, the β3-αC loop of MEK provides additional stabilizing interactions to the BRAF-bound AMP-PCP molecule. Utilizing an in vitro RAF dimerization assay, we find that MEK not only modulates RAF-RAF dimerization, it also enhances both the dimer-breaking effect ATP and the dimer-forming effect of paradoxical activator molecules, such as GDC-0879. Structural analysis reveals that all common oncogenic BRAF mutations alter key interactions with ATP that stabilize the inactive monomer conformation. We find ATP is unable to break RAF dimers containing Class I, II or III mutations, confirming these mutants counteract the inhibitory effects of ATP binding by lowering the threshold for RAF dimerization and thus pathway activation. We further find that oncogenic MEK mutants, in particular β3-αC loop deletions, favor RAF-RAF dimerization, even in the presence of ATP. Our study establishes a framework for rationalizing oncogenic BRAF mutations, and suggests that oncogenic MEK mutants can act, at least in part, by promoting RAF dimerization. This model provides a better understanding of activation mechanisms in the context of constantly evolving resistance mutations where oncogenesis continues to depend on the MAPK pathway. Citation Format: Timothy J. Wendorff, Jennifer Kung, Jawahar Sudhamsu. Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation [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 A036.

Abstract LB352: KRAS inhibition enhances immunogenicity of KRASG12C colorectal cancer

Abstract RAS mutations are the most common oncogenic mutations, present in >20% of human cancers. KRAS, the predominantly mutated isoform of RAS, is mutated in 40% of colorectal cancer (CRC), leading to constitutive activation of the MAPK (RAS/RAF) pathway and other downstream effectors. Due to the difficulty of targeting RAS, the first KRASG12C selective inhibitor has only been developed recently and approved by the FDA for non-small cell lung cancer. However, the inhibitor demonstrated much lower response rates in CRC (<20%) than other cancer types. Thus, developing effective therapies for KRAS mutant (KRASm) CRC still remains challenging. Oncogenic KRAS signaling has been shown to have immunosuppressive effects on tumor microenvironment, thus we hypothesized that KRAS inhibitor can indirectly enhance antitumor immunity, providing a rationale for their combination with immune checkpoint blockade. Indeed, using a syngeneic KRASG12C CRC mouse model with knockout of APC, TP53, SMAD4 and expressing KRASG12C, we observed significant increase in the percentage of CD3+CD8+ T cells as well as changes in myeloid populations in tumors treated with KRASG12C inhibitor compared with vehicle control. Combination of KRASG12C/PD-1 inhibition produced a more substantial and sustained reduction in tumor growth compared with KRAS or PD-1 inhibition alone. KRASG12C CRC mouse model represents a proper tool to study tumor cell-intrinsic mechanism of cooperativity between KRAS inhibition and immune response, since KRASG12C inhibitor targets KRASm tumor cells only without having direct effects on stromal and immune cells. Interestingly, we found tumor-intrinsic immune programs induction by KRAS/MAPK inhibition in KRASG12C CRC mouse models and patient-derived organoid models. We also found that KRASG12C inhibition upregulates T-cell recruiting chemokines CXCL9 and CXCL10 via IRF1 in KRASm tumor cells, which might be one of the potential mechanisms underlying enhanced infiltration of CD3+CD8+ T cells. These data suggest a link between KRAS inhibition and anti-tumor immunity, thus provide insights into novel combination strategies for KRASG12C colorectal cancers. Citation Format: Jun Tian, Ferran Fece de la Cruz, Kelly Burke, Tomonori Oka, Jacquelyn M. Curtis, Princy Sindurakar, Edmond Wong, Shadmehr Demehri, Arlene Sharpe, Ryan Corcoran. KRAS inhibition enhances immunogenicity of KRASG12C colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB352.

Biomarker-Driven Oncology Clinical Trials: Novel Designs in the Era of Precision Medicine

Oncology drug development historically has followed a path of sequential phase I, II, and III clinical trials using traditional trial designs, with the goal of achieving regulatory approval. These studies are often conducted with inclusion criteria that limit enrollment to a single tumor type or tumor site of origin, excluding other patients who might also respond. Increased use of precision medicine targeting biomarkers or specific oncogenic mutations has led to novel clinical trial designs that can evaluate these therapies in a less limited fashion. Master protocols such as basket trials, umbrella trials, and platform trials can, for example, evaluate histology-specific therapies targeting a common oncogenic mutation across multiple tumor types or screen for the presence of multiple different biomarkers rather than a single one. In other cases, they can lead to more rapid evaluation of a drug and evaluate targeted therapies in tumor types for which they are not yet currently indicated. As the use of complex biomarker-based master protocols increases, advanced practitioners must understand these novel trial designs, their advantages and disadvantages, and how their use may advance drug development and maximize the clinical benefits of molecular precision therapy.

Tumor MK2 transcript levels are associated with improved response to chemotherapy and patient survival in non-small cell lung cancer.

Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.