Development Of Inhibitors Research Articles

Abstract 682: Development of novel eIF4E inhibitors to potently and selectively suppress tumor growth across multiple indications

Abstract Despite considerable progress in treating cancer, drug resistance remains the primary hurdle to achieving cures in patients. Tumors often develop resistance through reactivation of the drug-targeted pathway, or activation of alternative parallel pathways, thereby maintaining the original downstream signaling effects. One of the furthest downstream effectors and critical nodes for many oncogenic signaling pathways is eukaryotic translation initiation factor 4E (eIF4E); the main regulator and rate limiting factor for protein synthesis. eIF4E is reactivated by many resistance mechanisms to promote translation of multiple pro-oncogenic factors including Cyclin D1/3, making it an attractive target to potentiate the anti-cancer activity of targeted therapies and to overcome drug resistance. Here, we present the development of novel, potent, and selective eIF4E inhibitors for use in both treatment-naïve and resistance settings across multiple tumor indications. To capitalize on the potential of targeting eIF4E, we developed a series of compounds with unique properties that maintain anti-tumor efficacy while minimizing toxicity. Our novel, selective, and potent oral eIF4E inhibitors (RBX-eIF4Ei) elicit a reversible, dose-dependent cell cycle arrest. Unlike targeting eIF4A, eIF4E inhibition selectively regulates translation of cancer-dependent pathway proteins instead of global protein synthesis, thus reducing on-target toxicity and increasing tolerability. Cellular profiling demonstrates RBX-eIF4Ei are highly selective, nanomolar inhibitors across many tumor types including, NSCLC, CRC, breast, and melanoma. Additionally, RBX-eIF4Ei demonstrate consistent efficacy across both sensitive and resistant cell lines, including intrinsic and acquired resistance models. Combining RBX-eIF4Ei with standard of care targeted therapies produces additive responses in both settings, suggesting its potential anti-neoplastic benefits post progression. In vivo, daily oral monotherapy treatment with RBX-EIF4Ei causes significant tumor growth inhibition across a variety of indications including BRAFmut CRC, BRAFmut melanoma and ER+ breast cancer, with minimal signs of toxicity. Intratumoral concentration of RBX-eIF4Ei correlate with significant tumor cell growth inhibition, as well as reductions in eIF4E target proteins, ODC1 and Cyclin D1. Combination studies with standard of care in both the treatment naïve and resistant settings are ongoing. Collectively, these data support the addition of RBX-eIF4Ei to standard of care in both the naïve and treatment resistance settings across a variety of indications including NSCLC, breast, CRC, and melanoma. IND enabling studies are planned, marking a significant step toward advancing these promising eIF4E inhibitors into clinical development. Citation Format: Matthew B. Friedersdorf, Devon R. Blake, Sarah E. Thompson, Roheeth Pavana, Ramamurty V. Changalvala, Andrew S. Truong, Krista Marran, Abha Bias, Jessica A. Sorrentino. Development of novel eIF4E inhibitors to potently and selectively suppress tumor growth across multiple indications [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 682.

Abstract 3164: The in vivo Hollow Fiber model is a valuable tool in drug development of selective KRas inhibitors

Abstract The proto-oncogene KRas is a well-described small GTPase that functions as a molecular switch for major physiological signaling pathways involved in cell proliferation, differentiation and survival. It has been shown that activating mutations in KRas are among the most common oncogenic drivers of tumorigenesis. Missense mutations of KRas result in constitutive activation due to impaired hydrolysis of GTP which enhances tumor-promoting downstream signaling pathways. Most KRas mutations are located in exon 2 or 3 including the most frequently altered glycine 12, which is present in most pancreatic cancers as well as in colorectal cancers and lung adenocarcinomas. A panel of cancer cell lines harboring different KRas mutations such as G12C, G12V or G12D was selected to test KRas inhibitors in several cellular assays: phosphorylation assays, 2D and 3D proliferation assays. Applying the cellular pERK AlphaLISA assay showed high selectivity of several inhibitors for specific KRas mutants, while RAF and MEK inhibitors did not show significant selectivity in the tested cancer cell lines. The KRas inhibitors were then examined in a cellular 2D and a 3D spheroid proliferation assay to determine the inhibitory effect on cell growth. These cellular assays revealed a high selectivity for a specific KRas mutant, which was even more pronounced in the 3D format than in the 2D setup. The transferability of the cellular data obtained to in vivo was tested in the Hollow Fiber mouse model. The Hollow Fiber model allows the simultaneous study of multiple cell lines implanted in separated drug-(but not cell-) permeable fibers in a single mouse with a study duration of only 16 days. AMG510 (Sotorasib), a drug approved for tumors with a G12C Kras mutation, and MRTX1133, which is currently in clinical phase and shows activity in tumors with a G12D Kras mutation, were screened for their inhibitory effect on the pancreatic tumor cells MiaPaCa-2 (G12C Kras mutation), AsPC-1 (G12D Kras mutation) and BxPC-3 (wt Kras) in the in vivo Hollow Fiber model in female NMRI nude mice. While the growth of BxPC-3 tumor cells was not affected by either inhibitor, the growth of AsPC-1 tumor cells in particular was selectively and significantly inhibited by MRTX1133. In summary, the in vivo Hollow Fiber model is a fast and cost-effective model that can play an prominent role in drug development as a link between in vitro and in vivo xenograft studies by providing rapid and transferable evidence for in vivo efficacy. Citation Format: Philipp Metzger, Claus-Werner Franzke, Ezgi Dikici, Gregor Sommerkamp, Franziska Fimm-Todt, Jan E. Ehlert, Cynthia Obodozie, Holger Weber. The in vivo Hollow Fiber model is a valuable tool in drug development of selective KRas inhibitors [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 3164.

Abstract 5902: Targeting MAPK-driven tumors via inhibition of MAP2K4

Abstract MAPK pathway activation is a feature of multiple tumor types. Drugging KRAS and BRAF, the two main oncogenic drivers in the MAPK pathway, has proven successful in the clinic. Inhibition of the downstream effectors, MEK and ERK, can also induce tumor regression. Despite this, many tumors are intrinsically resistant to MAPK pathway inhibitors, or acquire resistance under selective pressure to drug treatment. This creates a need for combination treatments to improve clinical responses. A synthetic lethal (SL) interaction between inhibition of the MAPK pathway and blockade of JNK-JUN signaling has recently been described1. Specifically, data from yeast genetics and CRISPR knockout experiments in human cells have identified MAP2K4 as a potential therapeutic target that could be combined with MAPK inhibitors. To date, however, no potent MAP2K4 inhibitors with in vitro and cellular selectivity against key anti-targets have been reported. Here, we describe the development of potent covalent inhibitors of MAP2K4 kinase activity. Biochemical and cell-based assays show that the compound(s) are selective for MAP2K4 versus anti-targets including MAP2K7 and ERK kinases. The combination of MAP2K4 and MEK/ERK inhibitors was effective in cell lines driven by MAPK signaling. These data provide the rationale for further development of MAP2K4 inhibitors to advance our understanding of this novel drug combination.

Abstract 4704: Biochemical and cell-based assay platforms for development of RAF inhibitors against human cancers

Abstract The RAF protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the RAS GTPases and as the initiating kinases in the ERK cascade. In human cancer, RAF activity is frequently dysregulated due to mutations in the RAF family members (ARAF, BRAF, and CRAF) or to alterations in upstream RAF regulators, including RAS and receptor tyrosine kinases. The first and second generations of RAF inhibitors have yielded dramatic responses in malignant melanomas containing BRAF mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In addition, cancers with hyperactive RAS exhibit intrinsic resistance to these drugs. In particular, issues related to the dimerization of the RAF kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. We have established biochemical HotSpotTM kinase assay, NanoBRETTM and NanoBITTM cell assay platforms for High Through Screening of kinase inhibitors. Here, we demonstrate that the 3rd generation of pan-RAF inhibitors LY3009120, LXH254, and Belvarafenib inhibit ARAF, BRAF, CRAF, BRAF(V600E), and CRAF(R391W) kinase activity in biochemical HotSpotTM assay. Our NanoBRETTM target engagement and NanoBITTM cellular assay data show that LY3009120, LXH254, and Belvarafenib bind to KRAS(G12C) primed BRAF and CRAF, and block BRAF and CRAF dimerization. Furthermore, our results show these inhibitors can block the downstream ERK phosphorylation in cellular HTRF assay and induce caspase-3/7 activation in Western blot assay in the triple negative breast cancer MDA-MB-231 cells. Taken together, our results indicate the biochemical HotSpotTM kinase activity assay, and NanoBRETTM target engagement and NanoBITTM cellular assays can serve as great platforms to facilitate RAF drug discovery against human cancers. Citation Format: Jianghong Wu, Yong Wan, Shuguang Liang, Peter Gallagher, Li Liang, Christian Loch, Haiching Ma. Biochemical and cell-based assay platforms for development of RAF inhibitors against human cancers [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 4704.

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 & 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 &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 4477: The development of a small molecule dual inhibitor of CXCR4 and Mcl-1 to target bortezomib resistant and stem-like cells in multiple myeloma

Abstract Multiple myeloma (MM) is a malignant plasma-cell illness defined by clonal growth of malignant plasma cells within the bone marrow (BM) microenvironment. Despite the availability of many therapeutic alternatives, MM remains consistently lethal, demanding the development of novel techniques. The intimate relationship between myeloma cells and the BM microenvironment is a distinguishing hallmark of MM. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 enhance the homing of MM cells to the BM, which has important functional implications. The BM microenvironment secretes a variety of hormones and cytokines that activate several signaling pathways, resulting in enhanced production of the anti-apoptotic Mcl-1 protein, boosting the proliferation, survival, and migration of MM cells and conferring resistance to standard chemotherapy. As a result, we created GA-A, a hybrid molecule in which we coupled an aralkyl carboxylic acid moiety, known to preferentially inhibit Mcl-1, to gambogic acid, which has previously been demonstrated to inhibit CXCR4 in MM. Our western blot has shown that this unique hybrid compound successfully suppressed CXCR4 and Mcl-1. Interestingly, using qPCR we found that CXCR4 was observed to be higher expressed in CD138- versus CD138+ cells, while GA-A inhibited CXCR4 in both subpopulations. In addition, using annexin-V flow cytometry staining we found that GA-A increased the apoptotic effect of bortezomib in MM stem-like cells while inhibiting the viability of bortezomib resistant cells. Using human cytokine array we found that GA-A activates some of proinflammatory cytokines that can activate the immune system such as GM-CSF, IFN gamma, IL-2 and IL-12. In summary, our findings indicate the successful creation of a novel dual CXCR4-Mcl1 inhibitor that demonstrates efficacy against the stem cell-like population and bortezomib resistant cells and enhances the effectiveness of bortezomib. Citation Format: Weam Othman Elbezanti, Omar S. Al-Odat, Subash C. Jonnalagadda, Tulin Budak-Alpdogan, Manoj K. Pandey. The development of a small molecule dual inhibitor of CXCR4 and Mcl-1 to target bortezomib resistant and stem-like cells in multiple myeloma [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 4477.

Abstract 4482: Development of small molecule inhibitors of lipocalin 2 for the treatment of aggressive cancers

Abstract Lipocalin-2 (LCN2), a secreted protein pivotal in iron homeostasis, immune responses, siderophore transport, and epithelial cell differentiation. LCN2 is highly abundant in aggressive forms of many cancers including breast, pancreatic, ovarian, and esophageal cancer. Elevated LCN2 levels in aggressive tumors correlate with heightened cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. After the protein folds it makes a barrel structure which encloses a three-calyx pocket that can bind and transport molecules. A first generation of small molecule inhibitors (SMIs) targeting LCN2 demonstrated efficacy in attenuating the proliferative capacity of Inflammatory breast cancer (IBC) cells. IBC stands as a rare and highly aggressive variant, accounting for 1 to 5% of all BC cases, with 20-30% presenting as de novo metastatic disease. In the previous studies the concentration of LCN2 inhibitors to reduce cell proliferation in 50% was around 10 µM. In pursuit of better LCN2 SMIs, we conducted in-silico analysis using a library of 369,000 ligands. Through the High-Performance Computer Facility at UPR Río Piedras, we employed AutoDock Vina for virtual screening, targeting the LCN2-calyx with Lys134 as the central coordinate. Docking protocols, predicting binding affinities, identified 8,162 out of 327,638 ligands that have been docked already with values below -9.6 kcal/mol for further consideration. The next phase involves subjecting these potential inhibitors to the SwissADME tool for a comprehensive evaluation of absorption, distribution, metabolism, and excretion parameters, as well as pharmacokinetics, drug-likeness, and medicinal chemistry considerations. The parameters to be evaluated are the Lipinski rules, which states that an orally active drug may have a maximum of one violation and the pann-assay interference compounds (PAINS) alerts tool, a source of false positives for biologically active compounds. The top candidates, exhibit binding affinities within the range of -11.5 to -12.2. After SwissADME filtering, we will re-rank the ligand list, identifying 30 commercially available candidates for subsequent cell viability assays in SUM149 (TNBC) and SUM190 (HER2+) cell lines. This multifaceted approach integrates computational methodologies with rigorous experimental validation, paving the way for potential therapeutic interventions in IBC treatment. Citation Format: Marienid Flores-Colón, Cristal A. Peña-Vázquez, Eliud Hernández-O'Farril, Luis E. Vázquez-Quiñones, Pablo E. Vivas-Mejía. Development of small molecule inhibitors of lipocalin 2 for the treatment of aggressive cancers [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 4482.

Abstract 1814: Development and evaluation of Hsp90β-selective inhibitors to overcome the safety challenges associated with pan-Hsp90 inhibitors

Abstract The 90 kD heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding of select proteins, many of which are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. More than 20 small-molecule inhibitors of Hsp90 have entered clinical trials for the treatment of cancer, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms (Hsp90a, Hsp90β, Grp94 and TRAP-1). Most of these clinical trials have failed as pan-Inhibition of Hsp90 appears to be detrimental with toxicities reported alongside induction of the pro-survival heat shock response, which leads to additional dosing challenges/toxicities. The cytosolic Hsp90 isoforms Hsp90a and Hsp90β are the major contributors to cancer growth/survival. It has been established that inhibition of Hsp90a is associated with cardiac toxicity, retinal toxicity, and induction of the heat shock response. Therefore, the development of Hsp90β -selective inhibitors represents an alternative approach for the treatment of cancer with limited side effects. Towards this goal, Hsp90β-selective inhibitors that exhibit >150-fold selectivity over Hsp90a have been developed, which exhibit efficacy against select cancers while overcoming the safety challenges associated with previous Hsp90 inhibitors. The results from such studies will be presented. Citation Format: Sanket Mishra, Tyelor Reynolds, Brian Blagg. Development and evaluation of Hsp90β-selective inhibitors to overcome the safety challenges associated with pan-Hsp90 inhibitors [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 1814.

Abstract 1946: Elucidating the mechanisms of acquired resistance to AMG510 in cancer models harboring KRAS G12C mutations

Abstract GTPase KRas (KRAS), a critical oncogene, is known for its frequent mutations across a wide array of human cancers, most notably in pancreatic, lung, and colorectal cancers. Recent advancements in the field have led to the successful development of numerous inhibitors targeting the mutant KRAS. Clinical application of KRAS G12C inhibitors, such as AMG510 and MTRX849, represents major breakthroughs in addressing previously 'undruggable' cancer targets. Nevertheless, a subset of patients initially responsive to these inhibitors eventually exhibit treatment resistance. Uncovering the underlying mechanism would help to develop novel therapies that overcome the acquired drug resistance. To elucidate the underlying mechanisms, we generated AMG510-resistant tumor models, namely AMG510-R-MIA PaCa-2 and AMG510-R CT26 KRAS G12C, by continuous administration of AMG510 to tumor-bearing mice. Upon comparison of sensitive parental tumors and resistant tumors, we found that highly expressed KRAS and constant activation of RAS-MAPK signaling pathway are critical for resistant tumor progression following AMG510 treatment. In addition, resistant tumor cells are responsible for the elevated secretion of CXCL2/3/5, which dramatically reconditions the tumor immune microenvironment and recruits immunosuppressive cells. In summary, the exploration of the resistance mechanism in AMG510-induced resistant models provides insight into the development of new-generation KRAS-G12C inhibitors and novel combinatorial therapies. Citation Format: Ting Ni, Tongxin Huo, Yawen Zhang, Jingjing Wang, Gaoxiang Liu, Wenting Shi, Qingyang Gu. Elucidating the mechanisms of acquired resistance to AMG510 in cancer models harboring KRAS G12C mutations [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 1946.

Abstract 4491: Development of IRE-1 inhibitors for B cell cancer therapy

Abstract In response to the endoplasmic reticulum (ER) stress, the IRE-1 kinase/RNase splices the mRNA of the XBP-1 gene, resulting in the spliced XBP-1 (XBP-1s) mRNA that encodes the functional XBP-1s transcription factor. XBP-1s is critically important for the growth and survival of B cell leukemia, lymphoma, and multiple myeloma. When compared with other inhibitors targeting IRE-1, the tricyclic chromenone-based inhibitors B-I09 and D-F07, prodrugs harboring an aldehyde-masking group, emerged as the very reliable inhibitors for potent suppression of the XBP-1s protein. Protection of the aldehyde as a 1,3-dioxane acetal led to strong fluorescence emitted by the coumarin chromophore, enabling both B-I09 and D-F07 to be tracked inside the cell. We installed a photolabile structural cage on the hydroxy group of D-F07 to generate PC-D-F07. Such a modification significantly stabilized the 1,3-dioxane acetal protecting group, allowing for specific stimulus-mediated control of the inhibitory activity. Upon photoactivation, the re-exposed hydroxy group triggered the aldehyde-protecting 1,3-dioxane acetal to slowly decompose, leading to the inhibition of the RNase activity of IRE-1. Since chemical modifications of the hydroxy group could be used to tune 1,3-dioxane prodrug stability, we installed reactive oxygen species (ROS)-sensitive boronate cage groups onto B-I09 (BC-B-I09) and D-F07 (BC-D-F07) to achieve stimuli-responsive activities and improve tumor-targeting efficiency based on that cancerous B cells indeed produced higher levels of ROS than normal B cells. These boronate caged compounds could be further combined with the FDA-approved Auranofin, a thioredoxin reductase inhibitor, to achieve synergistic cancer-killing effects. These results led us to further develop a novel prodrug, TC-D-F07, in which a thiol-reactive dinitrobenzenesulfonyl (Dns) cage was installed onto the hydroxy group of D-F07. The electron-withdrawing Dns group in TC-D-F07 similarly stabilized the neighboring 1,3-dioxane acetal, allowing for stimulus-mediated control of its inhibitory activity. TC-D-F07 exhibited high sensitivity to intracellular thiols produced by cancerous B cells. In addition, when TC-D-F07 was decomposed into D-F07, we observed that a dinitrophenyl cysteine adduct resulting from cleavage of the Dns group could induce the ER stress, causing cancerous B cells to increase the expression of XBP-1s. However, the accumulated levels of D-F07 from TC-D-F07 and its gradual decomposition into the active IRE-1 inhibitor could eventually deprive cancer cells of all XBP-1s, leading to more severe apoptosis in TC-D-F07-treated than in D-F07-treated cells. Thus, TC-D-F07 with both ER stress-inducing and XBP-1s-inhibiting activities represents an IRE-1-targeting caged prodrug that can be used to treat B cell cancer effectively. Citation Format: Andong Shao, Chih-Hang Anthony Tang, Juan R. Del Valle, Chih-Chi Andrew Hu. Development of IRE-1 inhibitors for B cell cancer therapy [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 4491.

Abstract 4583: Development of selective HDAC6 inhibitors to improve cancer immunotherapy

Abstract Histone Deacetylases (HDACs) are enzymes that modify histones and non-histone proteins. Our group has previously demonstrated that HDAC6 inhibitors (HDAC6is) modulate multiple immune pathways, including those that suppress the antitumoral M2 phenotype, leading to improved antitumor immunity. Despite the potential of HDAC6is, current inhibitors have poor selectivity and off-target effects and only work at micromolar concentrations. In this work, we designed a screening pipeline of HDAC6i, considering selectivity, potency, capacity to prevent the protumoral phenotype of macrophages, and low cytotoxicity on macrophages and other immune cells. This approach has not been tested before, as the standard screening method for antitumoral HDACis has focused on its cytotoxic effect on cancer cells. We initially designed in silico 980 compound derivatives from previously reported HDAC6i, such as SS208, Nexturastat A, and Suprastat. Twenty-four compounds with a high capacity to bind HDAC6 and chemical stability were screened by their ability to inhibit HDAC6 in cell-free conditions. Sixteen compounds demonstrated high potency and were further evaluated in murine macrophages by their cytotoxic effect using CellTox. The capacity to impede tubulin deacetylation was assessed by western blot and deacetylase activity by HDAC-Glo. The top six candidates were then evaluated by their selectivity compared to other HDAC members and by their effect on repressing the expression of protumoral M2 macrophages Arginase 1 and Fizz1, and activation of antitumoral markers TNFa and iNOS by western blot and qRT-PCR. The best three candidates were then evaluated at nanomolar concentrations to explore their immunomodulatory potential of M1 against M2 by performing antigen presentation (SIINFEKL presentation) and phagocytic capacity using microscopy. Two of these compounds, SM-06-09 and SM-05-947, will be further tested in syngeneic murine melanoma models for their ability in tumor regression and antitumor immunity. This study has yielded important insights into developing new and improved HDAC6is with minimal cytotoxicity, and we believe our work will help advance cancer research and ultimately lead to better patient outcomes. Citation Format: Nithya Gajendran, Manasa Suresh, David Quiceno, Xintang Li, Marie Durr, Karen Tan, Satish Kumar Reddy Noonepalle, Alejandro Villagra. Development of selective HDAC6 inhibitors to improve cancer immunotherapy [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 4583.

Abstract 538: Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models

Abstract Introduction: Poly (ADP-ribose) polymerases (PARPs) can recruit DNA repair effectors and cope with DNA single-strand breaks. For DNA double-strand breaks (DSB), there are two main repair mechanisms: non-homologous end joining repair (NHEJ) which is prone to error due to infidelity events, and BRCA1/2 mediated homologous recombination repair (HR) restoring the original sequence. Therefore, inhibition of PARPs may cause both single stranded break repair deficiency and HR deficiency in BRCA1/2 deficient patients, leading to cell death. The concept of “synthetic lethality” has led to the development of PARP inhibitors, including Olaparib, Niraparib and Rucaparib in clinical practice. Despite encouraging efficacy of these PARP inhibitors, acquired resistance, such as HR repair restoration, drug efflux pump upregulation and replication fork protection, has limited their clinical benefits. Therefore, to investigate novel therapeutic strategies which can overcome the acquired resistance, we established patient-derived xenograft (PDX) ovarian cancer models with PARP inhibitor-induced resistance. Methods: OV9402, an ovarian cancer PDX model with BRCA 1 c.3155del, was implanted subcutaneously and treated with Niraparib at 35 mg/kg, daily, for 129 days. Relapse tumors (tumor #1 and tumor #2) were isolated and inoculated into naïve mice. Then the 2nd round of treatment (Niraparib, 35 mg/kg, daily) was given when the mean tumor volume reached 140 mm3. The tumors showing no significant response to Niraparib were collected on either day 32 (#1 derived tumor model) and day 22 (#2 derived tumor model) after randomization and their gene changes were analyzed by RNA sequencing (RNA-seq). Results: Niraparib significantly inhibited OV9402 tumor growth, with complete regression observed in nine out of ten mice on day 54 after the 1st round of treatment. However, tumor regrowth was observed in two out of the nine mice from day 68 onwards. The relapsed tumors (tumor #1 and tumor #2) from these two mice were collected and re-inoculated into 5 naïve mice to establish tumor models for the 2nd round of Niraparib treatment, separately. For #1 derived tumor model, all the tumors presented complete resistance to the Niraparib treatment. RNA-seq data showed that the gene expression of ABCB1, which encodes for efflux pumps, was upregulated. In parallel, complete resistance to Niraparib was observed in all the tumors derived from 2# tumor bearing mice. The RNA-seq data demonstrated that an acquired secondary genetic deletion on BRCA1 genes, which restored the functional protein expression loss in the parental line and restored HR repair, was found in the tumors. Conclusion: Ovarian cancer PDX models that have effectively developed resistance to PARP inhibitors have the potential to serve as valuable preclinical tools to evaluate potential next-generation PARP inhibitors and innovative combination strategies. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Xiaobo Chen, Guo Sheng, Ludovic Bourre, Jingjing Wang. Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models [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 538.

Abstract 4698: Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery

Abstract Inhibitors of poly (ADP-ribose) polymerases (PARPs) have revolutionized cancer therapy by highlighting the potential of synthetic lethal drugs to target the DNA damage response (DDR) network. More recently, the development of PARG inhibitors was spurred by the discovery that inhibiting PARG (poly (ADP-ribose) glycohydrolase) results in the accumulation of poly ADP-ribose on DDR proteins and induces cell death. Adding PARG or PARP inhibitors to a cell of interest and quantifying the resulting levels of PARylation can provide valuable insight on compound membrane permeability and target engagement in a cellular context. Nevertheless, assessing PARylation (poly-ADP-ribosylation in which multiple ribose units are added in linear or branched forms) in cells is difficult compared to biochemical assays, impeding the progress of candidate drug development. A sandwich ELISA-based assay was developed to analyze the total protein PARylation present in cellular extracts. Experimental protocols were optimized to discern differences in cellular PARylation levels resulting from activating the DDR and from exposure to PARP inhibitors and/or PARG inhibitors. It is worth noting that our assay specifically detects PARylation, measuring the effects associated with PARP family members 1 to 5, as other family members primarily catalyze MARylation (mono-ADP ribosylation in which a single ADP-ribose unit is added). Titration of known PARP inhibitors and PARG inhibitors was performed to validate the assay. 1) PARG inhibitor PDD00017273, used in combination with DNA damaging agent H2O2, increased the level of cellular PARylation, exhibiting a similar IC50 value across the four cell lines tested. This is consistent with the notion that PARG is the major de-PARylating enzyme in most cells. The potency of other known PARG inhibitors PDD00017272 and PDD00017238 ranked the compounds as expected from published results (PDD00017272< PDD00017273< PDD00017238). 2) Titration of the three PARP1/2 inhibitors Talazoparib, Olaparib and AZD5305, in combination with a DNA damage agent and PDD00017273 inhibitor, shows a dose-dependent reduction in cellular PARylation in HEK293 cells, as expected since PARP1 is activated in response to DNA damage and this activation is blocked by the inhibitors (IC50 values, Olaparib = 0.013 µM, AZD5305 = 0.0024 µM, Talazoparib = 0.0016 µM). These IC50 are consistent with published values and underscore the usefulness of the assay in measuring dose response effects, allowing accurate determination of compound IC50 in living cells. In summary, our optimized experimental cell treatment protocols and ELISA-based assay allow quantification of total cellular PARylation and accurately rank candidate drugs targeting PAR erasers or PAR writers in a high-throughput format, using living cells. Citation Format: Caila Pilo, Mohammad Awad, Veronique T. Baron, Fernando Martins, Jonathan Mikolosko, Steven Plouffe, Zhihua Tao, Katarzyna Zientara-Rytter, Henry Zhu, Pavel Shashkin. Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery [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 4698.

Abstract 5079: Comparative studies of chemotherapy and immuno-oncology combinations in multiple mouse syngeneic tumor models

Abstract The development of immune checkpoint inhibitors (ICIs) and combinations of immuno-oncology therapies with standard-of-care (SoC) chemotherapeutic agents has shifted therapeutic paradigms for patients with multiple cancer indications. For many patients, these combinations have shown a beneficial effect on clinical outcomes and disease management. However, additional combination therapies are still needed. Preclinically, a comparison of responses from SoC chemotherapies and ICI combinations in multiple disease models may be helpful in identifying optimal treatment combinations in the clinic. Here we report the results of multiple SoC/ICI in vivo combination efficacy studies in a large panel of mouse syngeneic models spanning seven tissue origins: 1) Colon: CT26, MC38; 2) Breast: EMT6, 4T1; 3) Kidney: Renca; 4) Bladder: MB49, MBT2; 5) Skin: B16F10, CM3; 6) Lung: TC1, LL/2, M109; 7) Brain: GL261. The advantage of using syngeneic models is not limited to the rapid and reproducible tumor growth, but importantly that the animals also have an intact immune system and clinically relevant tumor microenvironment, which are required for ICI combination studies. We tested > 10 chemo SoC combinations with a murinized anti-PD-1 antibody (DX400) in 14 syngeneic mouse models. We found that multiple SoC regimens combined well with DX400 treatment, including FOLFIRI, FOLFOX (colon), Carboplatin/Paclitaxel, Carboplatin/Pemetrexed (lung), Capecitabine/Docetaxel, Cisplatin/Gemcitabine (bladder), Cisplatin/5-FU/or capecitabine, and Oxaliplatin/5-FU/or capecitabine (gastric). Specifically, we observed that the SOC/ICI combinations resulted in enhanced antitumor activities compared to single-agent treatments in many of the models. These data, particularly specific SoC combination regimens that combine well with ICIs in each disease, are useful for optimizing promising combination approaches for clinical development and will be presented here. Citation Format: Eunsil Park, Marlene Hinton, Mingmei Cai, Malgorzata A. Gil, Gustave Hebert, Long Cui, Doug Linn, Brian J. Long, Evan R. Barry. Comparative studies of chemotherapy and immuno-oncology combinations in multiple mouse syngeneic tumor models [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 5079.

Abstract 4066: Generation of tumor targeted self-assembling split IL-12 subunits for the treatment of cancer

Abstract A major milestone in immuno-oncology was the development of immune checkpoint inhibitors, resulting in significant benefits for numerous patients. However, many patients are not responsive, become refractory, or cannot tolerate the treatment due to toxicity or side effects, demonstrating the need for additional immunotherapies. Cytokines are a class of promising immunomodulatory proteins being explored as therapeutics, but their success has been limited due to their rapid clearance or pleiotropic properties. Classically known as ‘signal 3’ of an endogenous immune response, IL-12p70 is a heterodimeric cytokine comprising p35 and p40 subunits. When administered exogenously, IL-12 is a potent stimulator of the immune system and can have profound anti-tumor activity; however, the clinical use of IL-12 has been limited due to poor systemic tolerability. Therefore, developing an immunotherapy that can be systemically administered and improve the therapeutic index could capitalize on the potential clinical benefit of IL-12. Mural Oncology has developed an innovative approach to mitigate the toxicity of IL-12 by splitting the heterodimer into inactive p35 and p40 monomers. The individual subunits are separately fused to two non-competitive antibody fragments targeting a highly expressed tumor-associated antigen. The goal of this approach is to conditionally activate IL-12 preferentially in the tumor microenvironment (TME). This is achieved by sequential administration of the targeted subunits, which will drive assembly and activity of the IL-12 heterodimer primarily at the tumor site, reducing systemic exposure and thereby potentially reducing associated toxicities. To engineer the split IL-12 subunits, a structure-based rational design approach was used to remove the natural intra-molecular disulfide bond and generate variants that maintained affinity between the p35 and p40 monomers. The engineered subunits were characterized using analytical methods and screened for the pair with the strongest affinity. Additionally, the candidate pair was assessed for receptor binding and functional activity on human T cells through phosphorylation of STAT4 and secretion of IFN-γ. Pharmacokinetics and pharmacodynamics were evaluated in mouse models with and without tumors demonstrating that dual targeting led to an increase in tumor retention, rapid clearance from circulation, and dose dependent release of IFN-γ. The results thus far demonstrate that the innovative approach of tumor targeted self-assembling split IL-12 has the potential to enhance the overall therapeutic potential of this cytokine. A development candidate will be identified and moved into clinical testing in cancer patients to enable us to fully understand if this design approach holds true in the clinical setting. Citation Format: Joshua Heiber, Pinar Gurel, Robert G. Newman, Kristiana Dreaden, Yanchun Zhao, Chunhua Wang, Su-Ping Pearson, Jean Chamoun. Generation of tumor targeted self-assembling split IL-12 subunits for the treatment of 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 4066.

Abstract 5064: Wild type KRAS amplification as de novo oncogenic driver in non-small cell lung cancer

Abstract Introduction: Oncogenic KRAS mutations define subsets of non-small cell lung cancer (NSCLC) with unique clinicopathologic and genomic features, and the recent development of direct KRAS inhibitors has changed the treatment landscape of these patients (pts). However, whether KRAS amplification is oncogenic and defines unique subset of potentially targetable NSCLCs is currently unknown. Methods: Clinicopathologic and genomic data were abstracted from multiple independent cohorts of NSCLCs which underwent tumor genomic profiling at the Dana-Farber Cancer Institute (Cohort 1), AACR-GENIE v.13, TCGA, and other 212 studies (Cohort 2). Whole transcriptome sequencing and quantitative proteomics data from the The Cancer Genome Atlas (TCGA) and the Cancer Cell Lines Encyclopedia (CCLE) were used to correlate KRAS amplification with KRAS RNA levels and protein expression. KRAS doxycycline-inducible models were generated to determine in vitro and in vivo oncogenicity of wild-type KRAS overexpression. Results: Among 15,341 pts with NSCLC identified, KRAS amplification was identified in 355 (2.4%) cases. Compared to KRAS not amplified cases, pts with KRAS amplified NSCLC were more likely to be males (52.4% vs 45.6%, p=0.01), have a history of smoking (82.1% vs 64.2%, p<0.01), and higher median pack-years (35 vs 23.5, p<0.01). KRAS amplification was also associated with higher median aneuploidy levels (as fraction of genome altered) (30.4% vs 14.3%, p<0.01), tumor mutational burden (p<0.01), and increased median PD-L1 expression (20% vs 5%, p=0.01). KRAS amplified cases were enriched with KRAS, POLE2, SLC34A2 and PALB2 mutations, while EGFR mutations were mutually exclusive (Q<0.1). Among NSCLC samples with transcriptomic and proteomic profiling from the TCGA and CCLE cohorts, KRAS amplification was associated with significantly increased KRAS mRNA (p<0.01) and protein expression (p<0.01), compared to KRAS diploid samples. Among pts with clinical outcomes data available (N=9,335), median overall survival (OS) from the date of diagnosis was significantly shorter in pts with KRAS amplified vs KRAS diploid genotype in both KRAS mutant (HR: 0.71, P=0.02) and KRAS WT (HR: 0.74, P=0.02) NSCLCs. In KRAS doxycycline-inducible models (BEAS-2B cells) we demonstrated increased cell proliferation and MAPK activation upon doxycycline-induced KRAS expression. In vivo, sustained expression of KRAS in animals constantly fed with doxycycline-diet triggered tumor formation and development of metastasis, indicating that WT KRAS overexpression is oncogenic. Conclusion: KRAS amplification defines a novel subset of NSCLCs characterized by distinct clinicopathologic and genomic features, and worse survival. Developing novel therapeutic strategies to target KRAS amplification may improve outcomes in pts with NSCLC. Citation Format: Biagio Ricciuti, Sandra V. Michelina, Joao Alessi, Xinan Wang, Federica Pecci, Alessandro Di Federico, Malini Gandhi, Giuseppe Lamberti, Enrico Patrucco, Lynette Sholl, Andrew Aguirre, Chiara Ambrogio, Mark Magdi Awad. Wild type KRAS amplification as de novo oncogenic driver in non-small cell lung 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 5064.

Abstract 1970: Discovery and characterization of potent and selective HER2 exon20 insertion mutant inhibitors

Abstract Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for over 85% of all cases. Among NSCLC, approximately 2-4% of patients have mutations in the HER2, a member of the ErbB family of receptor tyrosine kinases and key proliferation driver, with exon 20 insertions making up about 90% of these mutations. HER2 exon mutations can have various alterations, and among them, the most common HER2 mutation is in-frame insertions in exon 20, especially the A775_G776insYVMA insertion/duplication. These mutations can affect the structure and function of the HER2 protein and may contribute to tumor development and growth. HER2 mutations have often shown resistance to EGFR tyrosine kinase inhibitors (TKIs), further highlighting the need for HER2-specific treatments. Also, inhibitors targeting HER2 mutations are selective, meaning they specifically target HER2 alterations without causing toxicities or affecting EGFRWT. Therefore, there is a need for the development of selective HER2 exon20 insertion mutant inhibitors. Herein, Hanmi has identified selective HER2 exon20 insertion mutant small molecule inhibitors, designed based on affinity and binding mode starting from a known crystal structure using GLIDE (Schrödinger), and the screening for the viability of cell lines including HER2A775_G776insYVMA, HER2G776delinsVC , HER2WT and EGFRWT was investigated. Hanmi's HER2 exon20 insertion mutant inhibitors selectively target mutant HER2 receptor tyrosine domains through shared binding, sparing EGFRWT and minimizing EGFR-related toxicities. With highly selectivity against EGFRWT and desirable pharmacokinetic properties, the Hanmi compounds inhibit HER2 mutant cells and HER2 mutant enzymes. Additionally, the compounds have an oral administration-compatible DMPK profile and demonstrated anti-tumor activity in HER2 mutant and HER2WT tumor xenograft mice. In conclusion, based on our study, Hanmi compounds, a novel HER2 exon20 insertion mutant inhibitors could be suggested as an appropriate therapeutic agent for inhibitory activity against HER2 exon20 insertion mutant NSCLC. Citation Format: Ho Yeon Nam, Sun Young Jang, Jiyoung Jeon, HyungSeok Yoo, Jooyun Byun, Gunwoo Lee, Youngjoo Lee, Yu-Yon Kim, Soye Jeon, Young Gil Ahn. Discovery and characterization of potent and selective HER2 exon20 insertion mutant inhibitors [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 1970.

Abstract 3227: Developing brain-penetrating MAT2A inhibitors for MTAP-deleted brain metastatic cancer and GBM

Abstract Background: Homozygous deletion of MTAP is one of the most frequent genetic alterations in various solid tumors including glioblastoma (GBM), mesothelioma, pancreatic cancer and lung cancer. While MTAP-deleted cancers are associated with a poor prognosis, there are no approved drugs for the treatment of patients with MTAP deficiency. MAT2A has emerged as a potential target for selective anticancer effects in MTAP-deleted cancers. However, development of brain-penetrating MAT2A inhibitors are yet to be reported in preclinical or clinical stages despite of high MTAP deletion fraction in GBM and brain metastatic cancers. Here, we present highly potent and selective MAT2A inhibitors capable of brain penetration for MTAP deleted cancer therapy. Methods: A MAT2A biochemical enzyme assay was performed with phosphonate colorimetric kit. To evaluate in vitro and in vivo activities of compounds, S-adenosylmethionine (SAM) and symmetrical dimethyl arginine (SDMA) levels were measured using LC-MS/MS and high-content screening systems. Cell growth inhibition assay was evaluated to confirm the selectivity in isogenic pairs of HCT116 WT and MTAP-deleted cells. In vivo efficacy was conducted using the MTAP-deleted HCT116 xenograft. Results: To develop novel brain penetrating scaffolds, we focused on interaction of hydrogen bond acceptor in the scaffolds with arginine residue Arg313 in MAT2A protein and the improvement of physicochemical properties, including logP and logBB. A series of MAT2A inhibitors significantly inhibited MAT2A enzymatic activity, resulting in the reduction of intracellular SAM level, the direct product of MAT2A. These compounds showed highly potent inhibition of SDMA and cell proliferation in MTAP-deleted cells, while displaying minimal effect on MTAP-WT cells, showing >100-fold selectivity towards MTAP-deletion over MTAP-WT. Furthermore, anti-proliferative activity of MAT2A inhibitors were observed in MTAP-deleted cancer cells across various cancer types, including GBM, breast cancer, and non-small cell lung cancer. Remarkably, mouse pharmacokinetic study revealed high level of plasma and brain exposure with outstanding brain-to-plasma ratio (> 0.75). Robust tumor growth inhibition as well as reduction in tumor SAM levels were observed as monotherapy in an MTAP-deleted HCT116 xenograft model. Conclusion: We have identified novel brain-penetrating MAT2A inhibitors displaying encouraging in vitro and in vivo pharmacological profiles, indicating the potential use of these compounds as therapeutic agents for MTAP deleted brain metastatic cancer and GBM. Citation Format: Soyoung Ki, Jinhee Kim, Jungtae Na, Ilkyoo Koh, Hyunho Cho, Ho Yeon Lee, Hoiyun Jung, Gyeonghi Cho, Mijin Moon, Yongje Shin, Sook-Kyung Park. Developing brain-penetrating MAT2A inhibitors for MTAP-deleted brain metastatic cancer and GBM [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 3227.

Abstract 649: SOX11 regulates BCR signaling via the PAX5/CD19 axis in MCL

Abstract Background: Mantle cell lymphoma (MCL) is an incurable lymphoid neoplasm, comprising about 6% of all non-Hodgkin lymphoma (NHL). The transcription factor SOX11 is overexpressed in the majority of MCL patients and linked to a more aggressive disease course and poorer clinical outcomes. In MCL, naïve B cells experience continuous proliferation because of the dysregulation of the B-cell receptor (BCR) pathway. Our previously published data (Kuo et. al., Blood 2018) identified that SOX11 aberrantly upregulated BCR signaling in transgenic mouse models. The current study describes the exact mechanism by which SOX11 regulates the BCR pathway using a combination of functional and pharmacological approaches. Methods: To investigate the alterations in signaling pathways regulated by SOX11, we utilized a small molecule SOX11 inhibitor (Jatiani et. al., CCR 2021), performed CRISPR/CAS9 knockout of SOX11, 3X FLAG TAG overexpression, as well as conducted western blot and flow-cytometry experiments. Results: Our prior published results (Kuo et. al., Oncogene 2015), as well as others (Vegliante et. al., Blood 2013), have shown that PAX5 is a direct target of SOX11. We confirmed PAX5 downregulation after SOX11 depletion in three human MCL cell lines (JEKO, MINO, Z138) using CRISPR-CAS9. Decrease of PAX5 expression was associated with reduction in CD19 expression and a concurrent reduction in phospho-BTK (Bruton tyrosine kinase). Conversely, overexpression of SOX11 could rescue PAX5/CD19 expression and restore p-BTK levels. Furthermore, the treatment of SOX11 expressing cells with small molecule SOX11-specific inhibitors resulted in a reduction in PAX5, CD19 and BCR activation (p-BTK). Conclusion: Taken together, these findings delineate PAX5 as the direct target and PAX5/CD19 axis as the downstream pathway by which SOX11 regulates B-cell receptor (BCR) signaling in MCL. Notably, our treatment using a SOX11 inhibitor successfully replicated the effects observed with genetic knockdown. In conclusion, our findings provide valuable insights into the molecular mechanisms by which SOX11 contributes to MCL pathogenesis. The further development of pharmacological inhibitors targeting SOX11 holds promise as a beneficial therapeutic strategy for MCL patients, as these will block signaling upstream of current resistance mechanisms. Citation Format: Rudra Prasad Dutta, Heng-Huan Lee, Violetta V Leshchenko, Ravi Prakash Shukla, Yang Liu, Jian Jin, Michael Wang, Samir Parekh. SOX11 regulates BCR signaling via the PAX5/CD19 axis in MCL [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 649.

Abstract 4548: Discovering novel ALK-lung carcinoma therapeutic strategies by identifying combinations with Alectinib from ALK-positive lung carcinoma cell lines

Abstract Background: The therapeutic landscape for chromosomally rearranged anaplastic lymphoma kinase (ALK) has focused on the development of Tyrosine-kinase inhibitors (TKI) that specifically target ALK activity. However, as patients progress on first- and second line TKIs, over 50% of resistance is due to non-mutational resistance via compensatory pathways, also known as ALK-non dominant resistance. Alterations in other drivers such as EGFR, KRAS, MET account for some of the resistance mechanisms. Thus, here we explore the therapeutic activity of drug combinations that pair an ALK inhibitor with a drug that targets a "second driver". We describe our high-throughput drug screening platform that combines Alectinib, a selective blocker of ALK, with a panel of hundreds of chemical compounds curated to be active against a diverse array of pathways involved in cellular functions dysregulated in cancer. In this work, we identified several synergistic combinations that further increased the sensitivity of five ALK-positive lung cancer cell lines (DFCI 032, NCI-H3122, NCI-H2228, SNU2535, SNU324) to Alectinib in comparison to single treatment with the final goal of targeting resistance, before it emerges. Methods: ALK-positive cancer cell sensitivity to different compounds was assessed by generating the IC50 curves in the presence and absence of Alectinib. Cells were characterized by bulk-RNAseq, bulk-DNA seq, Reverse-phase protein array, and Mass spectrometry to identify perturbed RNA pathways, DNA mutations, and proteome alterations. Results: In this study, we present for the first time, a group of compounds, such as Gilteritinib, Trametinib, Dinaciclib, WYE-125132, Cenisertib, SN-38 which exhibit the capacity to induce cytotoxic effects in ALK-positive lung cancer cells, when combined with Alectinib. Our findings demonstrate a novel sensitivity of ALK driven by non-conventional parallel signaling, apart from conventional targets previously described, such as phosphoinositol-3kinase (PI3K)/Akt, signal transducer and activator of transcription 3 (STAT3), and extracellular signal-regulated kinases (ERK). Conclusions: Through the integration of different multiomic approaches on five ALK-positive lung cancer cell lines, we have pinpointed a novel role of MAPK, CDK, and mTOR, pathways and their regulators, as a promising avenue for advancing treatment strategies for ALK-positive lung cancer. These findings, along with the identification of several other significant targetable elements illustrate a new approach towards the discovery of new drug combinations and targetable pathways to tackle ALK TKI resistance in ALK-positive patients before it emerges. We have previously generated Alectinib-resistant models and are in the process of screening to identify any resistant pathways that may align with those identified in the sensitive lines. Citation Format: Hamadi Madhi, Habib Serhan, Nathalie M. Vandecan, Zhaoping Qin, Albert Liu, Aaron Udager, Sofia D. Merajver, Matthew B. Soellner, Nathan Merrill. Discovering novel ALK-lung carcinoma therapeutic strategies by identifying combinations with Alectinib from ALK-positive lung carcinoma cell lines [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 4548.