Molecular mechanisms underlying sex-specific differences in cancer incidence and therapy responses are under intense investigation. Here, we report sex-biased functions of Yap1 in multiple cancer types in human and mouse. Through integrated multi-omics analyses, we demonstrate that Yap1 deletion significantly extends survival in male but not female Sonic Hedgehog (SHH) medulloblastomas (MB) models. While Yap1 is required to maintain stem-like cells in both sexes, Yap1 plays a more critical role in immune evasion in males. Mechanistically, YAP1 is essential for activating Cd276/B7-H3 expression to mediate CD8+ T cell suppression in males. Consistently, CD276 inhibition extends survival in male but not female SHH MB. Moreover, in vivo targets of YAP1 stratify survival in male but not female patients with medulloblastoma, glioblastoma, mesothelioma, and lung cancer. This study provides evidence for sex-biased functions of Yap1 and CD276 in MB immune suppression and highlights the importance of biological sex in cancer:immune interactions.

The second Paediatric Therapeutic Development Workshop focused on medulloblastoma. Between 60-70% of patients with medulloblastoma survive, but survivors have significant long-term side effects, and the highest-risk groups have a probability of survival <10%. Thus, the unmet need is to develop therapeutics targeting specific vulnerabilities in medulloblastoma including poor prognosis disease groups (SHH-medulloblastoma, MYCN amplified or TP53 mutated; and Group 3 medulloblastoma, c-MYC amplified) and developing less-toxic therapies for good prognosis disease (WNT-medulloblastoma). The Workshop concluded that (i) targeting SRC by a degrader is a high priority, (ii) inhibition of c-MYC and MYCN tumour-relevant functions for poor prognosis groups is a priority, (iii) targeting WNT-medulloblastoma via a radiolabelled theranostic antibody is an innovative approach for good prognosis tumours to further reduce toxicity, and (iv) B7-H3 has many advantages for CAR T-cell and ADC-based approaches. Based on currently available evidence, combinations of central nervous system penetrant selective PARP-1, CHK1/2 or CDK9 inhibitors with an ATR inhibitor could potentially be evaluated in early-phase trials for high-risk patients; however, these combinations require robust evaluation in pre-clinical models first. Early-phase clinical studies should be international, have novel designs to address small patient numbers and based on an understanding of biology with correlative biological studies. Both developing therapeutics targeting specific vulnerabilities in medulloblastoma and evaluating combinations of existing medicinal products are required to improve outcome and reduce long term sequalae.

The molecular mechanisms driving sex disparities in cancer prevalence, progression, and treatment outcomes represent a crucial yet understudied area with significant implications for cancer therapy. Medulloblastoma (MB), exhibits a pronounced sex bias in incidence and survival rates, with males experiencing higher incidence and poorer prognoses compared to females. This study focused on the role of Yap1 in Sonic Hedgehog medulloblastomas (SHH MBs), which revealed an unanticipated sex-biased role of Yap1 in MB immune evasion. Hippo/Yap pathway is one of the top 10 most dysregulated pathways in human cancer. Our in vivo studies uncovered that Yap1 deletion in a spontaneous SHH MB model significantly extended survival, providing genetic evidence that Yap1 is a critical oncogene in MB formation. Notably, Yap1 deletion/inhibition benefitted males, but not females, revealing its sex-biased function for the first time. Integrated multi-omics analyses showed Yap1 is essential for maintaining MB stem/progenitor cells by activating stemness genes (Sox2) and repressing differentiation genes (Neurod1, Zic1/2) in both sexes. However, Yap1 plays a more pivotal role in immune evasion in males, particularly by regulating the immune checkpoint molecule; Cd276/B7-H3. In males, either CD276 blockade or Yap1 deletion reverses T cell suppression. In contrast, in females, CD276 blockade or Yap1 deletion is not sufficient to overcome T cell suppression, and it requires both interventions to activate T cells. Furthermore, our newly discovered transcriptional target gene signatures of YAP1 predict survival in male but not female patients across multiple human cancers, indicating a highly conserved mechanism across species and cancer types. This study highlights sex-specific differences in tumor:immune interactions downstream of YAP1/CD276. In addition, it reveals a novel sex-biased role of YAP1 transcriptional programs, providing molecular entry points for future research on sex differences in immune response. These results underscore the importance of considering sex as a major variable in mechanistic and therapeutic studies involving Yap1 and CD276, with broader implications for targeted cancer therapies. Nourhan Abdelfattah, Sivaraman Natarajan, Han Nhat Tran, Thomas Wong, Jose Maldonado, Rachael McMinimy, Hannah Borland, Shu-hsia Chen, Fernando Camargo, James Olson, Joshy George, Kyuson Yun. Yap1 and CD276/B7-H3: key players in Sex-biased SHH medulloblastoma immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6446.

The adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is effective in B cell malignancies. However, the persistence of cancer cells with low levels or complete absence of the target antigen, thereby evading detection by CAR T cells, leads to relapse. These evasion mechanisms highlight the need for receptors with enhanced sensitivity and multispecificity. We introduce a synthetic chimeric T cell receptor (ChTCR) that confers superior antigen sensitivity compared with CARS and previous hybrid TCR designs and is readily adapted for bispecific targeting. ChTCRs replicate the structure of natural TCRs, form classical immune synapses and demonstrate TCR-like signaling. T cells expressing bispecific ChTCRs (Bi-ChTCRs) are more effective than bispecific CAR T cells in eradicating tumors with heterogeneous antigen expression in vivo in female mice. The Bi-ChTCR architecture is resilient and can be designed to target pairs of B cell and multiple myeloma antigens. These findings provide a widely applicable strategy to combat tumor heterogeneity and prevent relapse. Simon, Bugos and colleagues report the design and characterization of monospecific and bispecific synthetic chimeric T cell receptors, conferring increased antigen sensitivity and antitumor activity in B cell malignancies and multiple myeloma models.

Background:The Children's Oncology Group (COG) study ACNS0332 examined the effect of adding carboplatin and isotretinoin to high-risk medulloblastoma therapy. Isotretinoin arms were closed early due to futility, but the effect of carboplatin was shown to vary by individual medulloblastoma subgroups. Because isotretinoin arms were closed before subgroup classification was available, a differential effect of isotretinoin among various subgroups was not examined. Here, we conduct a secondary analysis of ACNS0332 data examining the effect of isotretinoin on event-free survival (EFS) among individual medulloblastoma subgroups. Methods:Among 261 patients enrolled in ACNS0332, a subgroup was evaluable in 231 patients. Fisher's exact tests and chi-square tests were used to compare distributions of categorical variables among patients with and without exposure to isotretinoin. EFS for subgroups was estimated, and the log-rank test was used to examine differences in outcome distributions among patient groups. Results:Among 231 evaluable patients, 85 were randomized to isotretinoin, 85 were randomized to no isotretinoin, and 61 received no isotretinoin without randomization. All 4 medulloblastoma groups were identified: Randomization to isotretinoin was not associated with any difference in EFS in patients with group 3 (n = 79, P = .87), group 4 (n = 101, P = .53), SHH (n = 37, P = .69) or WNT (n = 14, P = 1) medulloblastoma. Conclusions:This study confirms that isotretinoin in addition to radiation and chemotherapy did not improve EFS in pediatric high-risk medulloblastoma regardless of molecular subgroup.

Background Despite intensive therapies, outcomes for high-risk pediatric brain tumors (PBTs) remain dismal, prompting the search for novel treatments. DNA methyltransferase inhibitors (DNMTi) have been shown to prime tumors to improve response to checkpoint inhibition. The aim of this study was to investigate the potential of decitabine (DAC), in combination with a PD-1 inhibitor, to improve survival in pediatric high-risk brain tumor models. Methods Analysis of human PBT datasets was performed to determine gene expression levels of immune cell associated markers. Tumor response to DAC, with or without a PD-1 inhibitor, was tested in murine models representing H3-wildtype diffuse intrinsic pontine glioma (DIPG), H3K27-mutant diffuse midline glioma (DMG), atypical teratoid rhabdoid tumor (ATRT), and medulloblastoma (MB). CyTOF analysis of allograft tumors was performed to characterize changes within the tumor microenvironment. Results Analysis of PBT subtypes revealed heterogeneous expression of immune cell markers, checkpoint receptors, and MHC molecules. DAC treatment decreased DNA methylation and increased neoantigen expression in human and mouse tumor cells. DAC alone or in combination with a PD-1 inhibitor resulted in prolonged survival in syngeneic mouse models of DIPG and ATRT but not DMG and MB models. CyTOF analysis of mouse tumors revealed changes in local immune cell infiltration upon combination treatment. Conclusions DAC in combination with a PD-1 inhibitor can alter the immune microenvironment in mouse tumor models. Changes were observed in H3-wildtype DIPG and ATRT models, suggesting that certain tumor subtypes may respond to checkpoint blockade after immune augmentation with DNMTi. Key Points 1. PBTs show heterogenous expression of immune cell infiltrates 2. DAC or DAC plus a PD-1 inhibitor shows extension of survival in H3-wildtype DIPG and ATRT mouse models 3. Myeloid-derived suppressor cell abundance could be a major contributing factor to treatment response Importance of the Study Children with high-risk PBTs face dismal outcomes. Immune checkpoint inhibitor (ICI) successes have been demonstrated in a variety of adult malignancies; however, such beneficial outcomes have not been realized in PBTs. Here we investigate single and combination treatment of DNMTi and PD-1 checkpoint inhibition in syngeneic mouse models of high-risk PBTs. Our results suggest that some H3-wildtype DIPG and ATRT tumor types may be responsive to checkpoint therapy post immunomodulation and warrant further investigation. ### Competing Interest Statement The authors have declared no competing interest. Stand Up to Cancer Catalyst grant Cure Starts Now Foundation, https://ror.org/04edq9h05 The Canadian Institute of Health Research

Abstract BACKGROUND Medulloblastoma has been redefined into four molecular subgroups (WNT, SHH, Group 3, and Group 4), with distinct prognoses and responses to treatment. The ACNS0332 trial investigated the impact of carboplatin and isotretinoin in high-risk medulloblastoma finding a differential effect of carboplatin by molecular subgroup. An interim futility analysis showed no benefit from isotretinoin, but did not evaluate results by molecular subgroup in the primary analysis. We performed a secondary analysis to examine whether the effect of isotretinoin on event-free survival (EFS) differed by molecular subgroup. METHODS Evaluable patients with known molecular subgroups were analyzed. Molecular subgroup analysis was conducted retrospectively in 231 patients at a central laboratory by methylation array. Kaplan-Meier survival analysis compared EFS (time from enrollment to disease progression or recurrence, second malignancy, death from any cause, or to date of last follow-up for patients without events). RESULTS Based on data from the 2014 interim analysis, 164 patients were eligible for secondary analyses. Group 3 (n=56, 34.1%) and Group 4 (n=75, 45.7%) were the most common groups. SHH (n=22, 13.4%) and WNT (n=11, 6.7%) groups were less common. There was no difference in isotretinoin (p=0.87) or carboplatin (p=0.49) randomization by subgroup. Race was well-balanced with expected differences in age (p=0.017) and sex (p=0.016) among molecular subgroups. Based on the interim analysis, there was no evidence of a difference in EFS by isotretinoin randomization (p=0.34; one-sided log-rank test stratified by carboplatin randomization). Based on secondary analysis data, there was no difference in EFS between patients receiving isotretinoin or no additional therapy in any molecular subgroup: Group 3 (p=0.68), Group 4 (p=0.55), SHH (p=0.68), WNT (p=1) based on two-sided log-rank tests. CONCLUSIONS Despite differential efficacy of carboplatin among molecular subgroups, no difference was found in EFS between patients receiving isotretinoin and those who did not, regardless of molecular subgroup.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen -presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR) -engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR -T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

Pediatric high-grade gliomas are highly invasive and essentially incurable. Glioma cells migrate between neurons and glia, along axon tracts, and through extracellular matrix surrounding blood vessels and underlying the pia. Mechanisms that allow adaptation to such complex environments are poorly understood. N-cadherin is highly expressed in pediatric gliomas and associated with shorter survival. We found that intercellular homotypic N-cadherin interactions differentially regulate glioma migration according to the microenvironment, stimulating migration on cultured neurons or astrocytes but inhibiting invasion into reconstituted or astrocyte-deposited extracellular matrix. N-cadherin localizes to filamentous connections between migrating leader cells but to epithelial-like junctions between followers. Leader cells have more surface and recycling N-cadherin, increased YAP1/TAZ signaling, and increased proliferation relative to followers. YAP1/TAZ signaling is dynamically regulated as leaders and followers change position, leading to altered N-cadherin levels and organization. Together, the results suggest that pediatric glioma cells adapt to different microenvironments by regulating N-cadherin dynamics and cell-cell contacts.

Background The paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor receptor 2 (HER2) is an attractive candidate for CAR T-cell therapy in humans but has the potential for eliciting on-target off-tumor toxicity. We developed an immunocompetent tumor model of CAR T-cell therapy targeting murine HER2 (mHER2) and examined the effect of CAR affinity, T-cell dose, and lymphodepletion on safety and efficacy.Methods Antibodies specific for mHER2 were generated, screened for affinity and specificity, tested for immunohistochemical staining of HER2 on normal tissues, and used for HER2-targeted CAR design. CAR candidates were evaluated for T-cell surface expression and the ability to induce T-cell proliferation, cytokine production, and cytotoxicity when transduced T cells were co-cultured with mHER2+ tumor cells in vitro. Safety and efficacy of various HER2 CARs was evaluated in two tumor models and normal non-tumor-bearing mice.Results Mice express HER2 in the same epithelial tissues as humans, rendering these tissues vulnerable to recognition by systemically administered HER2 CAR T cells. CAR T cells designed with single-chain variable fragment (scFvs) that have high-affinity for HER2 infiltrated and caused toxicity to normal HER2-positive tissues but exhibited poor infiltration into tumors and antitumor activity. In contrast, CAR T cells designed with an scFv with low-affinity for HER2 infiltrated HER2-positive tumors and controlled tumor growth without toxicity. Toxicity mediated by high-affinity CAR T cells was independent of tumor burden and correlated with proliferation of CAR T cells post infusion.Conclusions Our findings illustrate the disadvantage of high-affinity CARs for targets such as HER2 that are expressed on normal tissues. The use of low-affinity HER2 CARs can safely regress tumors identifying a potential path for therapy of solid tumors that exhibit high levels of HER2.

Abstract Unveiling the molecular mechanisms underlying sex-specific differences in cancer initiation, progression and treatment outcomes will provide novel insights that will advance cancer research and clinical care. This study highlights an unexpected, sex-biased role of the oncogene Yap1 in SHH medulloblastoma (MB). We discovered that Yap1 deletion in SmoM2-driven SHH MB significantly prolongs survival in male but not female mice. Using an integrated multi-omics approach, we show that YAP1 promotes cancer stem cell maintenance through concurrent transcriptional activation of stemness genes, such as Sox2, and repression of differentiation genes such as NeuroD1 and Zic1/2. Interestingly, while Yap1 is essential for maintaining cancer stem cells in both sexes, it plays a more critical role in immune evasion in males. Specifically, YAP1 regulates the expression of an immune checkpoint molecule Cd276 to suppress T cell function. Blocking CD276 or deleting Yap1 is sufficient to significantly reverse T cell suppression in males but not females. Furthermore, YAP1 direct targets of transcriptional regulation, including CD276, predicts survival in male but not female patients across multiple human cancers, suggesting that our findings have broader implications beyond medulloblastomas and is conserved across species. This study provides compelling evidence for male-biased susceptibility to Yap1 inhibition and uncovers the YAP1-CD276 axis as a sexually diverse pathway in T cell suppression in tumors. Citation Format: Nourhan Abdelfattah, Sivaraman Natarajan, Han Nhat Tran, Jose Maldonado, Rachael McMinimy, Hannah Borland, Shu-hsia Chen, Fernando Camargo, James Olson, Joshy George, Kyuson Yun. Sex-specific differences in Yap1 and Cd276 function and expression regulate medulloblastoma progression and immune evasion [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 3037.

Abstract Ewing sarcoma (EwS) is an aggressive bone and soft tissue tumor and EwS patients often succumb to their disease years after initial treatment due to relapses at metastatic sites. Novel therapeutic strategies are needed that can successfully eliminate microscopic residual disease foci that persist at the end of primary therapy. Extracellular matrix (ECM) proteins in the tumor microenvironment (TME) provide critical pro-survival and pro-invasion signals to tumor cells. Our published and unpublished data show that the glycoprotein tenascin-C (TNC) is enriched in EwS metastases and that TNC and other pro-tumorigenic ECM proteins are deposited by subpopulations of CAF-like tumor cells that are activated in response to tumor and TME-derived TGF-beta ligands. TNC is abundantly produced by multiple human tumors of epithelial and non-epithelial lineage but is otherwise rarely expressed outside of development and wound healing. Importantly, TNC is specifically enriched in metastatic lesions where it has been implicated as a mediator of metastatic competence and treatment resistance. In the current work we have tested whether the TNC-rich TME of disseminated EwS tumor foci could be leveraged to direct therapies to sites of residual micrometastatic disease. To achieve this, we generated monovalent and bivalent anti-human TNC VHHs (hTNC-VHH) using a mammalian expression platform. Camelid-derived hTNC-VHH sequences were sourced from published literature. VHHs are single domain heavy chain only antibody fragments with higher stability and tissue penetration than conventional monoclonal antibodies. Using EwS tumor spheroids in collagen-rich 3D culture, hTNC-VHH penetrated dense ECM matrices and bound through multiple cell layers in under 10 minutes. hTNC-VHH accumulated in TNC-positive but not TNC-knockout EwS spheroids and were retained beyond 96 hours. To assess their potential to drive protein and immune therapies to TNC-rich TMEs, we fused hTNC-VHH to immune modulating CD3- and CD28-activating VHHs. These hTNC-VHH-CD3/CD28-conjugates promoted immune cell activation and tumor cell death in PBMC-tumor co-culture assays as determined by CD25 flow cytometry and fluorescence microscopy, respectively. To assess in vivo localization, fluorescently-tagged hTNC-VHH were intravenously injected into mice that had been xenografted with EwS cells. Tissue microscopy confirmed selective binding of hTNC-VHH to small lung and liver EwS micrometastases in less than 4 hours. VHHs were not retained in non-tumor bearing organs including the brain and heart or in tumor-free lung and liver regions. Experiments are ongoing to assess anti-tumor efficacy and pharmacodynamics of hTNC-VHH conjugates in EwS xenografts. Together these findings suggest that the ECM-remodeling properties of CAF-like EwS cells can be exploited to recruit novel ECM-targeting protein therapeutics to micrometastases. If successful, this innovative approach could eradicate microscopic residual disease and prevent metastatic EwS recurrence. Citation Format: Emma D. Wrenn, Jason P. Price, Raymond O. Ruff, Nicolas M. Garcia, James M. Olson, Elizabeth R. Lawlor. Leveraging ECM deposition by CAF-like Ewing sarcoma tumor cells to target micrometastases with matrix-binding VHHs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C050.

The expression of a synthetic chimeric antigen receptor (CAR) to redirect antigen specificity of T cells is transforming the treatment of hematological malignancies and autoimmune diseases [1-7]. In cancer, durable efficacy is frequently limited by the escape of tumors that express low levels or lack the target antigen [8-12]. These clinical results emphasize the need for immune receptors that combine high sensitivity and multispecificity to improve outcomes. Current mono- and bispecific CARs do not faithfully recapitulate T cell receptor (TCR) function and require high antigen levels on tumor cells for recognition [13-17]. Here, we describe a novel synthetic chimeric TCR (ChTCR) that exhibits superior antigen sensitivity and is readily adapted for bispecific targeting. Bispecific ChTCRs mimic TCR structure, form classical immune synapses, and exhibit TCR-like proximal signaling. T cells expressing Bi-ChTCRs more effectively eliminated tumors with heterogeneous antigen expression in vivo compared to T cells expressing optimized bispecific CARs. The Bi-ChTCR architecture is resilient and can be designed to target multiple B cell lineage and multiple myeloma antigens. Our findings identify a broadly applicable approach for engineering T cells to target hematologic malignancies with heterogeneous antigen expression, thereby overcoming the most frequent mechanism of relapse after current CAR T therapies.

Epstein-Barr virus (EBV) is associated with several malignancies, neurodegenerative disorders and is the causative agent of infectious mononucleosis. A vaccine that prevents EBV-driven morbidity and mortality remains an unmet need. EBV is orally transmitted, infecting both B cells and epithelial cells. Several virally encoded proteins are involved in entry. The gH/gL glycoprotein complex is essential for infectivity irrespective of cell type, while gp42 is essential for infection of B cells. gp350 promotes viral attachment by binding to CD21 or CD35 and is the most abundant glycoprotein on the virion. gH/gL, gp42 and gp350, are known targets of neutralizing antibodies and therefore relevant immunogens for vaccine development. Here, we developed and optimized the delivery of several alphavirus-derived replicon RNA (repRNA) vaccine candidates encoding gH/gL, gH/gL/gp42 or gp350 delivered by a cationic nanocarrier termed LION™. The lead candidate, encoding full-length gH/gL, elicited high titers of neutralizing antibodies that persisted for at least 8 months and a vaccine-specific CD8+ T cell response. Transfer of vaccine-elicited IgG protected humanized mice from EBV-driven tumor formation and death following high-dose viral challenge. These data demonstrate that LION/repRNA-gH/gL is an ideal candidate vaccine for preventing EBV infection and/or related malignancies in humans.

Brain tumors are the most common solid tumor in children and the leading cause of cancer-related deaths. Over the last few years, improvements have been made in the diagnosis and treatment of children with Central Nervous System tumors. Unfortunately, for many patients with high-grade tumors, the overall prognosis remains poor. Lower survival rates are partly attributed to the lack of efficacious therapies. The advent and success of immune checkpoint inhibitors (ICIs) in adults have sparked interest in investigating the utility of these therapies alone or in combination with other drug treatments in pediatric patients. However, to achieve improved clinical outcomes, the establishment and selection of relevant and robust preclinical pediatric high-grade brain tumor models is imperative. Here, we review the information that influenced our model selection as we embarked on an international collaborative study to test ICIs in combination with epigenetic modifying agents to enhance adaptive immunity to treat pediatric brain tumors. We also share challenges that we faced and potential solutions.

Abstract The molecular mechanism governing sex disparities in cancer prevalence, progression, and treatment outcomes represents an important yet understudied area of research with significant implications for cancer treatment. Medulloblastoma (MB), the most prevalent pediatric brain malignancy, exhibits a notable sex bias in incidence and survival rates. Males have a higher incidence rate across all age groups and have worse prognoses than their females for age groups over three years old. Here, we report an unanticipated sex-biased role of the oncogene Yap1 in SHH medulloblastoma. Our results reveal that Yap1 deletion significantly prolongs survival in male, but not female mice with SmoM2-driven SHH MB. Employing an integrated multi-omics approach, we demonstrate that YAP1 is required to maintain cancer stem cells simultaneously activating stemness genes (such as Sox2) and repressing differentiation genes (such as Neurod1 and Zic1/2) in both sexes. In contrast, Yap1 plays a more critical role in immune evasion in males than in females. Specifically, YAP1 regulates the expression of the immune checkpoint molecule Cd276/B7-H3, which suppresses cytotoxic T cell function. In males, CD276 blockade or Yap1 deletion in MB cells is enough to reverse T cell suppression effectively. In females, neither CD276 blockade nor Yap1 deletion alone in MB cells can reverse T cell suppression. Furthermore, YAP1’s direct transcriptional target gene signatures, including CD276, predict survival across multiple human cancers in male patients. These findings suggest broader implications of our findings beyond medulloblastoma and indicate a highly conserved mechanism across species. Our study provides compelling evidence for the male-biased efficacy of Yap1 or CD276 inhibitors and presents novel insights into sex-specific mechanisms of cancer immune evasion.

Introduction. B-myeloid mixed-phenotype acute leukemia (B-MPAL) is a high-risk leukemia subtype presenting with both lymphoid (e.g. CD19) and myeloid (e.g. CD33) surface antigens. Cure rates in children and adolescents remain lower than for almost all other acute lymphoblastic leukemia (ALL) subtypes, with overall survival ranging from 75-80%. Outcomes for adults are notably poorer, with overall survival ranging from 20-50% for patients over 40 years of age. Induction therapies range from those used for ALL versus those used for acute myeloid leukemia (AML) with a paucity of information from randomized clinical trials due to the uncommon nature of this subtype. B-Myeloid MPAL cells frequently express both CD19 and CD33 while non-neoplastic human hematopoietic cells don't express both targets, creating an opportunity to selectively target MPAL cells with minimal off tumor toxicity. We hypothesize that a multispecific T-cell engager antibody (MTE) engineered to preferentially bind cells expressing CD19 AND CD33 will selectively kill MPAL cells compared to a bi-specific antibody that requires CD19 OR CD33 expression. We thus engineer MTEs that bind both CD19 and CD33 on MPAL cells and CD3 on T cells. We further fine-tune the affinity of the CD19 and CD33 binders so that the MTE binds poorly to single positive (SP) normal cells that express either CD19 or CD33 but binds well to double positive (DP) leukemia cells. For this we take advantage of both affinity and avidity, the latter of which occurs only when the MTE interacts with both CD19 and CD33 expressed on the same cell. Such therapies could be used to induce deep remissions, improve outcomes when integrated with chemotherapy, and/or serve as a bridge to hematopoietic cell transplantation. Blinatumomab, a bispecific antibody recognizing only CD19, has been a successful addition to treating both childhood and adult ALL. However, patients require months of intravenous immunoglobulin replacement therapy due to the consequential depletion of healthy B-cells, which share CD19 as a critical surface antigen with most B-cell ALL. The new therapeutic strategy we are developing will improve safety compared to currently available immunotherapies. Methods. We first identified that CD19 and CD33 are appropriate targets for B-Myeloid MPAL after analyzing aggregated transcriptome data for 31 MPAL patients. Next, we successfully designed and produced 30 tri-specific antibodies targeting CD19/CD33/CD3. All the binders were selected because they are either components of FDA approved therapeutics, have gone through human clinical trials, or have been extensively studied to de-risk safety and immunogenicity concerns. Our MTE framework is derived from the asymmetric “knob into holes” human IgG1 scaffold incorporating effector silencing mutations. Our in vitro pipeline to test those antibodies included binding assays as well as T-cell cytotoxic assays using cell lines expressing both targets, only one, or none. Top candidates were further tested for efficacy in in vivo mouse models. We use a luciferase labeled JIH-5 cell line that innately expresses CD19 and CD33, xenografted into NSG-SGM3 immunocompromised mice. We dosed engrafted mice with both MTE and human T-cells from a healthy donor for repeated weekly cycles. We followed disease progression every week using IVIS imaging. Results. Our preliminary data demonstrate our initial in vitro success in targeting CD19/CD33/CD3 antigens as well as dramatic improvement of specificity by generating lower affinity variants of known CD19 and CD33 binders. For multiple candidates we observed a much lower cytotoxicity for SP cells, with up to a 7 log difference in IC50. This difference in IC50 potentially expands the therapeutic window because DP cells are specifically targeted for lysis while single positive cells are spared. In addition, we present initial pre-clinical in vivo data for our top candidates in xenograft mouse models, showing successful control of disease in engrafted mice at treatment doses that are well tolerated. Conclusions. Taking advantage of the unique features of MPAL we developed a novel immunotherapy with improved half-life, potency, selectivity, and safety. This work serves as a proof of concept that the combinatorial use of binders with the proper affinity for their respective targets allow for the generation of highly specific immunotherapies with minimal cytotoxicity for normal cells.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. To identify genes differentially required for the viability of GBM stem-like cells (GSCs), we performed functional genomic lethality screens comparing GSCs and control human neural stem cells. Among top scoring hits in a subset of GBM cells was the F-box-containing gene FBXO42 , which was also essential in ∼15% of cell lines derived from a broad range of cancers. Mechanistic studies revealed that, in sensitive cells, FBXO42 activity prevents chromosome alignment defects, mitotic cell cycle arrest, and cell death. The cell cycle arrest, but not the cell death, triggered by FBXO42 inactivation could be suppressed by brief exposure to a chemical inhibitor of Mps1, a key spindle assembly checkpoint (SAC) kinase. FBXO42 ’s cancer-essential function requires its F-box and Kelch domains, which are necessary for FBXO42’s substrate recognition and targeting by SCF ubiquitin ligase complex. However, none of FBXO42’s previously proposed targets, including ING4, p53, and RBPJ, were responsible for the observed phenotypes. Instead, our results suggest that FBOX42 activity suppresses the accumulation of one or more proteins that perturb chromosome-microtubule dynamics in cancer cells, which, in turn, leads to induction of the SAC and cell death.