Genetic, epigenetic, and transcriptomic analyses have stratified medulloblastoma (MB) into four canonical subgroups of Wingless Type (WNT), Sonic Hedgehog (SHH), and Group 3 and Group 4, with distinct patient profiles and prognoses. Recent classification strategies have also considered combining Group 3 and Group 4 tumors into a Non-WNT/Non-SHH subgroup to account for biological overlap and heterogeneity. Using high-dimensional gene expression data from 487 pediatric and young adult patients and over twenty-one thousand transcripts, this study explores which genes can improve prognostic accuracy for survival while accounting for molecular stratification, histological subtype, key oncogenic drivers (MYC and MYCN amplification), and established clinical covariates, including age group (< 3 vs. 3–21 years) and metastatic status. We then develop a multi-stage framework for identifying prognostic genes and evaluating modern survival modeling strategies. In the first stage, gene screening was performed using Benjamini–Hochberg adjusted Cox regression across false discovery rate (FDR) thresholds from 1

Abstract Introduction Despite the decrease in childhood cancer mortality, a cure for certain tumors remains elusive. The survival rates for high-grade medulloblastoma are disappointing, pointing to the immediate need for creative therapeutic strategies. In the tumor microenvironment (TME), suppressor cells inhibit the immune system, which includes myeloid-derived suppressor cells- a heterogeneous population of immature myeloid cells that play a role in cancer progression and metastasis. Two subsets of these cells, granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs), promote this immunosuppressive milieu by inhibiting anti-tumor T cell activity. Methods Monocytes were co-cultured with HTB-186 cells in a transwell system to generate tumor-educated MDSCs. CFSE-labeled T cells were co-cultured with MDSCs and stimulated with anti-CD3/CD28 beads. Proliferation was analyzed by flow cytometry (FACSCanto, FlowJo v10). Data were analyzed using GraphPad Prism 8.0 (t-test, mean ± SEM). Results Our study revealed that M-MDSCs serve as a biomarker for high-grade, metastatic pediatric cancers, including medulloblastoma. Our findings suggest that high concentrations of the soluble factors GM-CSF and IL-6, which are produced by HTB-186 medulloblastoma cells, lead to the induction of medulloblastoma M-MDSCs. M-MDSCs from medulloblastomas are highly immunosuppressive and exert their suppressive function by inducing reactive oxygen species (ROS). Our data indicate that GM-CSF from medulloblastoma cells induces ROS production in M-MDSCs via the coordinated activation of p47phox, a critical subunit of the NOX2 NAPDH oxidase, by STAT3 and NF-κB, resulting in inhibition of T cells leading to immune suppression in the TME. Conclusion In conclusion, our study identifies M-MDSCs as key drivers of immune suppression in high-grade pediatric medulloblastoma. These findings highlight M-MDSCs as promising therapeutic targets to overcome immune suppression and improve outcomes in pediatric brain tumors. Funding Source PON2 728 2300001864 and PON2728 2400001585 to KY Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

IntroductionClear Cell Renal cell carcinoma (ccRCC) is the most common form of kidney cancer. The VHL tumor suppressor is lost or mutated in the majority of ccRCC and hereditary mutations in VHL predispose patients to ccRCC. However, another tumor suppressor, RASSF1A is inactivated at even higher levels in ccRCC. The majority of ccRCC lose function of both proteins. This suggests there may be some functional link between them. We found that the RASSF1A and VHL proteins can form a complex, suggesting they may act in concert to suppress the tumorigenic phenotype.MethodsWe generated two matched sets of Renal cancer tumor cell systems where we added back or knocked down both genes. We also made compound transgenic mice with induced, dual inactivation.ResultsIn the add-back system we found that the proteins cooperated to suppress 3D cell growth and sensitized cells to apoptosis inducing agents. In the knockdown system, we found the double loss cooperated to promote growth in 3D. In cell line xenografts, we found that the loss of VHL alone was insufficient to promote tumorigenesis, but it acted as an accelerant on tumor growth by RASSF1A deficient cells, which showed enhanced vascularization differential deregulation of HIF1α compared to HIF2α.DiscussionThis is the first report of an interaction between RASSF1A and VHL and may explain why RASSF1A is so frequently inactivated in Renal cancer. However, further studies in transgenic mice showed that dual inactivation of the tumor suppressors did not result in the formation of tumors or macrocysts. Thus, additional genetic lesions are likely required for the development of ccRCC.

RAS oncoproteins are the most frequently activated oncoproteins in cancer. Development of direct RAS inhibitors has proved technically challenging and has had limited success in the clinic. Those RAS inhibitors that have been approved tend to suffer from resistance development. Consequently, many attempts have focused on inhibiting RAS indirectly by targeting its immediate downstream effectors. RAS binds and activates three main effector classes to drive transformation: RAF kinases, phosphoinositide 3 (PI-3) kinase and Ras-like (RAL) small GTPases (RALGEF) exchange factors. Multiple FDA-approved inhibitors for RAF and PI-3 kinase exist. So far, they have proved to be of limited effectiveness in patients. However, no inhibitors of the RALGEF effectors with demonstrated antitumor activity have been reported. This is despite the considerable body of evidence supporting a critical role for the RALGEF/RAL pathway in facilitating the in vivo transforming effects of activated RAS. Here, we describe the first small molecule pan-RALGEF inhibitor. We show the inhibitor specifically suppresses RAS/RAL signaling and exhibits antitumor effects in xenograft experiments, including a patient-derived xenograft (pdx) model. This first-in-class compound may lead to the development of more effective therapies for a broad range of RAS-driven tumors.

Combination treatment with immunotherapy plus concurrent chemotherapy (cisplatin or carboplatin) is now part of the standard of care for many patients with non-small cell lung cancer (NSCLC). Although chemotherapy is known to enhance the efficacy of immunotherapies, whether chemotherapy can also have paradoxical negative effects that diminish immunotherapy efficacy is currently unknown. Our data indicate that cisplatin, a chemotherapy commonly used to treat NSCLC, induces prostaglandin E2 (PGE2) production in tumor cells; PGE2 leads to upregulation of CD73 enzyme on the surface of monocytic myeloid-derived suppressor cells (M-MDSCs). Our data show that CD73+ M-MDSCs catalyzes the production of extracellular adenosine that then inhibits the activation of effector T cells within the tumor microenvironment (TME) and thus limits the efficacy of chemo-immunotherapy. The production of PGE2 from lung tumor cells is regulated by a signaling pathway involving fatty acids, NF-κB, CREB, and COX-2. To deplete the intra-tumoral adenosine, we have explored the use of an FDA approved drug, [PEGylated]-Adenosine Deaminase (PEG-ADA). Our data show that depletion of adenosine with PEG-ADA induces anti-tumor immunity and sensitize NSCLC to immunotherapy. These findings reveal a novel immunosuppressive mechanism driven by cisplatin-induced PGE2 production and suggest that targeting adenosine metabolism is a novel strategy to improve the effectiveness of chemo-immunotherapy in NSCLC. NIH/NCI R01 (1R01CA272772-01A1) to KY Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Although mortality from childhood cancer is decreasing, a cure remains elusive for certain tumors. Survival rates for patients with high-grade medulloblastoma are dismal. Thus, the need for new therapeutic strategies remains an urgent unmet need. Within the tumor microenvironment (TME), suppressor cells curb the immune system, including myeloid-derived suppressor cells—a heterogeneous population of immature myeloid cells that play a role in cancer progression and metastasis. Two subsets of these cells—granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs)—promote this immunosuppressive milieu by inhibiting anti-tumor T cell activity. In our study, we identified M-MDSCs as a biomarker for high-grade, metastatic pediatric cancers including medulloblastoma. Our data indicated that medulloblastoma M-MDSCs are induced by high levels of the soluble factors GM-CSF and IL-6 produced by HTB-186 medulloblastoma cells. M-MDSCs from medulloblastomas are highly immunosuppressive and exert their suppressive function by inducing reactive oxygen species (ROS). Our data indicate that GM-CSF from medulloblastoma cells induces ROS production in M-MDSCs via the coordinated activation of p47phox, a critical subunit of the NOX2 NAPDH oxidase, by STAT3 and NF-κB, resulting in inhibition of T cells leading to immune suppression in the TME. These results pave the way to new therapeutic approaches for targeting immunosuppression in pediatric medulloblastoma.

NORE1A (RASSF5) is a tumor suppressor that is frequently down-regulated in liver tumors. It is an upstream component of the HIPPO pathway, a key regulator of liver development and metabolism. HIPPO disruption can lead to the development of MASLD/MASH. While studying the phenotype of NORE1A knockout mice, we noticed that they exhibit no overt liver tumor phenotype, but have a strong propensity to develop fatty livers characteristic of MASLD/MASH. Additionally, knockdown of NORE1A in liver cells upregulates sterol regulator element binding protein 1 (SREBP1), whose deregulation is central to the development MASLD. Examination of primary human MASLD samples showed an inverse correlation between the expression of NORE1A protein and TAZ, a downstream effector of the HIPPO pathway. Thus, loss of NORE1A expression may contribute to the development of MASLD/MASH in humans and NORE1A knockout mice may provide a new MASLD/MASH model that more accurately mimics the human disease.

Abstract The RAS oncoprotein has not traditionally been considered as an important driver of breast cancer due to the paucity of RAS mutations in this disease. However, RAS signaling pathways are frequently hyper-activated in breast cancers and genetic/epigenetic inactivation of RAS negative regulators (GAPs) is common. This is particularly true for Luminal B breast cancer. Luminal breast cancer makes up the majority of Breast Cancers, ~ 65% of cases. About 2/3 of these are classed as Luminal A and about 1/3 being the Luminal B class. Although current therapeutic options for luminal A (surgery, endocrine therapy) can be reasonably effective, Luminal B tumors are much more dangerous. These tumors are less sensitive to therapy in the first place and have a high frequency of relapse. Moreover, Luminal B tumors tend to occur more frequently in younger women and have a higher incidence of metastasis. One recent analysis has shown that Luminal B cancers have little better overall survival rates than the notorious triple negative breast cancers. Therefore, better therapies for Luminal B breast cancer are urgently required. One approach may be to target the hyper-active wild-type RAS oncoprotein that drives many of these Luminal B tumors. We have used in silico library screening followed by Medicinal Chemistry optimization to develop a series of novel small molecules that bind to all three main wild type RAS proteins and inhibit RAS function. We have validated the agents by using Microscale Thermophoresis binding procedures to quantify recombinant protein interaction. We used 3D growth inhibition and protein-based RAS signaling assays to quantify the biological action of the agents. Finally, we validated one of the more effective agents in vivo against a Luminal B cell line xenograft. This may be the first example of an effective anti-RAS therapy in breast cancer. It serves as proof of principal for the use of ant-RAS drugs in Luminal B disease. As basal breast cancers often exhibit loss of function of the RAS GAP called NF1, this strategy may have applications beyond Luminal B disease. Citation Format: Geoff Clark, Raphael Jigo, Howard Donninger, Joe Burlison, Mike Sabo, tariq Arshad, John Trent. Pan-RAS inhibitors to treat luminal B beast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B020.

Abstract RAS oncogenes are frequently activated by mutations in human cancer, where they act as driving elements of the disease. Mutation rates can range from ~25% of Non Small Cell Lung cancers to over 90% of pancreatic cancers. RAS proteins can also be hyper-activated to drive cancer at a significant frequency in the absence of RAS gene mutations when their protein regulatory systems are damaged. Thus, RAS may be the most frequently activated oncoprotein in cancer. We have developed a series of novel, direct, RAS inhibitors with a unique binding site on RAS. The binding of the agents alters the structure of the RAS effector loop and blocks the association with RAS downstream effectors. We used in silico library screening followed by Medicinal Chemistry optimization to develop the compounds. We have validated the agents using Microscale Thermophoresis and NMR to quantify binding to recombinant RAS protein. We have used mutant and wild type RAS driven tumor cell lines in 3D growth and protein-based RAS signaling assays to quantify and characterize the action of the family of inhibitors. We have used xenograft assays to demonstrate compound anti-tumor activity in RAS driven cell lines and pdx systems. We have identified compounds that can bind mutant and wild type K, H, N and M-RAS. We can suppress the association of mutant and wild type RAS protein with its effector RAF-1 in treated cells. The compounds can suppress 3D growth of mutant or wild type RAS driven tumor cell lines and repress tumor development in vivo. The compound family bind to a different site than either clinical agent AMG-510 (K-RAS G12C specific) or MRTX-1133 (K-RAS G12D specific). They exhibit distinct RAS signal inhibition patterns compared to these agents. They can also co-operate with the clinical agents and reduce drug resistance effects. Citation Format: Tariq Arshad, Howard Donninger, Goeff Clarke, Joe Burlison, Rob Monsen, Mike Sabo. In vivo and in vitro experience with novel direct Pan-RAS 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 3320.

NORE1A (RASSF5) is a tumor suppressor of the RASSF family that is often down-regulated in human tumors. NORE1A has multiple roles in controlling cellular homeostasis, one of them being regulating levels of β-catenin by binding and modulating the ubiquitin ligase substrate recognition factor β-TrCP. β-catenin is a major executor of the Wnt pathway. The ubiquitin SCF-β-TrCP ligase complex acts on a phospho-degron site in β-catenin that can be phosphorylated by GSK-3β. We now show that in addition to binding β-TrCP, NORE1A also promotes the phosphorylation of the β-catenin phospho-degron by complexing with the kinase GSK-3β. Indeed, NORE1A enhances the formation of a GSK-3β/β-TrCP complex. A structural mutant of NORE1A that retains β-TrCP binding but will no longer interact with GSK-3β inhibits the β-catenin degrading action of NORE1A. The GSK-3β interaction with NORE1A plays an important role in the biology of NORE1A as a GSK-3β inhibitor blocks NORE1A induced senescence. Thus, we identify a new role for the tumor suppressor NORE1A: The regulation of GSK-3β. GSK-3β has many other substrates including multiple transcription factors and co-activators such as p53 and the Hippo component TAZ. The work implies that NORE1A may be able to influence all of them via this new kinase scaffolding interaction.

RAS oncogenes are frequently activated by mutations in pancreatic and lung cancer, where they appear to act as driving mutations. This study examines the activity of a series of novel direct RAS inhibitors with a predicted unique interaction region. We have used in silico library screening followed by Medicinal Chemistry optimization to develop the compounds. We have validated the agents using Microscale Thermophoresis to quantify binding to recombinant RAS protein as well as NMR analysis of the drug/RAS complex. We used 3D growth inhibition assays in mutant RAS cell lines and protein-based RAS signaling assays to quantify and characterize the action of the family of inhibitors. We have identified compounds that bind wild type K-RAS and H-RAS but exhibit preferential binding to the K-RAS-G12D and KRAS-G12C mutants. We can suppress the association of mutant RAS protein with its effector RAF-1 in treated cells. We also observe suppression of mutant RAS signaling and inhibition of 3D cell growth of mutant RAS cell lines with the agents. As the compound family are predicted to bind to a different site than either AMG-510 (K-RAS G12C specific) or MRTX-1133 (K-RAS G12D specific), agents we have also tested co-operative activity with these drugs. Our compounds enhanced the effects of MRTX-1133 against K-RAS G12D cells and had a similar effect on AMG-510 in K-RAS G12C cell lines. These agents may serve as novel anti-RAS therapeutics and may have potential to enhance the activity or suppress resistance to AMG-510 and MRTX-1133. Citation Format: Geoff Clarke, Tariq Arshad, Howard Donninger, Becca von Baby, Rachel Ferrill, Mike Sabo, Joe Burlison, John Trent. Novel direct RAS inhibitors for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B006.

Supplementary Table 1 from NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

Despite the remarkable outcome of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC), a large number of patients remain unresponsive to this therapy. A key factor of the unresponsiveness to ICIs is intrinsic therapeutic resistance where monocytic myeloid-derived suppressor cells (M-MDSCs) contribute significantly via suppressing CD8 +T cell mediated anti-tumor response. Our results showed that in tumor microenvironment (TME), tumor cells derived PGE 2; a prostaglandin that directly induces CD73 (an ecto-nucleotidase that converts AMP to immunosuppressive adenosine) expression in M-MDSCs; consequently, CD73 expressing M-MDSCs suppress T cells via adenosine thus confer resistance to ICIs. In addition, we have shown that the depletion of adenosine via PEGylated Adenosine Deaminase (PEG-ADA) can reinvigorate CD8 +T cells, ultimately enhance the anti-tumor immunity.

4155 Background: Mutant-activated RAS genes are the most frequently mutated gene family associated with cancer (almost 30% of all cancers contain a mutant RAS gene). KRAS is the predominant isoform mutated in cancer and is the isoform exclusively mutated in pancreatic ductal carcinoma (PDAC). Since almost all PDAC cases harbor a mutant RAS, it is arguably the most RAS-addicted tumor type. There is now considerable evidence implicating mutant KRAS as a driver of PDAC. Recently, several mutant KRAS-targeted therapies (sotorasib and adagrasib) have been developed and show promise in PDAC patients. We have developed a direct inhibitor of RAS with a predicted unique interaction region capable of directly binding to wild-type H- and K-RAS, but which shows preferential binding for KRAS G12D and G12C mutants. This novel inhibitor disrupts the RAS effector domain and blocks the ability of RAS to signal through its effectors. Methods: We used in silico virtual library screening to identify an initial candidate inhibitor which was effective at inhibiting the 3D growth of PDAC cells without affecting their growth in 2D. Subsequent iterative rounds of medicinal chemistry was then performed to identify a series of derivatives with enhanced activity, as determined by 3D growth inhibition assays and effects on Ras signaling, as determined by Western blot analysis of phosphor-ERK, phosphor-Akt and activation of Ral A. Results: Our series of RAS inhibitors effectively block PDAC cell growth in 3D without impacting their 2D proliferation and suppress the interaction of KRAS with its effector cRAF. The inhibitors also effectively inhibit RAS signaling in mutant RAS containing PDAC cells. Since our novel compounds are predicted to bind to RAS at a different site to either AMG-510 (G12C specific inhibitor) and MRTX-1133 (G12D specific inhibitor), we tested the co-operativity of our compounds with these existing agents. Our compounds enhanced the anti-proliferative effects of both MRTX-113 and AMG-510 in mutant KRAS G12D and G12C PDAC cells, respectively. Conclusions: We have developed a series of novel RAS inhibitors that directly bind preferentially to mutant KRAS that may serve as new mutant KRAS-targeted therapeutics, and that may also have the potential to enhance the efficacy or suppress the resistance of AMG-510 and MRTX-1133.

Abstract Since RAS mutations are rare in Breast Cancer, RAS has not traditionally been considered as an important driver of this disease. However, RAS signaling pathways are frequently hyper-activated in breast cancers and genetic/epigenetic inactivation of RAS negative regulators (GAPs) is common. This is particularly true for Luminal B breast cancer. Luminal breast cancer makes up the majority of Breast Cancers, ~ 65% of cases. The Luminal B form makes up around 1/3 of these. Although current therapeutic options for luminal A (surgery, endocrine therapy) can be reasonably effective, Luminal B tumors are much more dangerous. These tumors are less sensitive to therapy in the first place and have a high frequency of relapse. Moreover, Luminal B tumors tend to occur more frequently in younger women and have a higher incidence of metastasis. One recent analysis has shown that Luminal B cancers have little better overall survival rates than the notorious triple negative breast cancers. Therefore, better therapies for Luminal B breast cancer are urgently required. One approach may be to target the hyper-active wild-type RAS oncoprotein that drives many of these tumors. We have in used in silico library screening followed by Medicinal Chemistry optimization to develop a series of novel small molecules that bind to all three main RAS proteins and block RAS function. We have validated the agents by using Microscale Thermophoresis binding procedures to quantify recombinant protein interaction. We used 3D growth inhibition and protein-based RAS signaling assays to quantify the biological action of the agents. Finally, we validated one of the more effective agents in vivo against a Luminal B cell line xenograft. This may be the first example of an effective anti-RAS therapy in breast cancer. It serves as proof of principal for the use of ant-RAS drugs in Luminal B disease. As basal breast cancers often exhibit loss of function of the RAS GAP called NF1, this strategy may have applications beyond Luminal B disease. Citation Format: Geoff Clarke, Howard Donninger, Raphael Jigo, Tariq Arshad, Becca von Baby, Rachel Ferrill, Mike Sabo, Joe Burlison, John Trent. RAS inhibitors to treat luminal B breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B005.