Survival In Murine Model Research Articles

IMMU-09. REVERSING SYSTEMIC T CELL DYSFUNCTION TO LICENSE IMMUNOTHERAPY IN INTRACRANIAL TUMORS

Abstract Intracranial tumors present unique challenges for immunotherapy. These can include both local and systemic modes of immune suppression, the mechanistic underpinnings of which are incompletely understood. Our work reveals that intracranial tumors elicit systemic increases in circulating catecholamine levels, and that this chronic sympathetic hyperactivity results in T cell dysfunction that impedes immunotherapeutic efficacy. We show that treatment with b-adrenergic blockade can partially overcome the negative impacts of chronic sympathetic hyperactivity by increasing NF-kB activity in immune cells, restoring T cell polyfunctionality, favorably modifying the tumor microenvironment, and extending survival in murine models of glioblastoma treated with immune-based therapies. Furthermore, we demonstrate that extended survival is also observed in glioblastoma patients receiving b-adrenergic blockade, as well as in patients with melanoma and lung cancer brain metastases who received b-blockade alongside concomitant immune checkpoint inhibition. Importantly, while local sympathetic hyperactivity in the tumor microenvironment and b-blockade also impact the anti-tumor immune response in the context of extracranial disease, these effects are markedly more pronounced in intracranial disease. Taken together, these data reveal that sympathetic hyperactivity facilitates systemic immune dysfunction in the setting of intracranial tumors and highlight a novel role for the application of b-adrenergic blockade to license immunotherapy in intracranial malignancies.

ATRT-15. COMBINING THE PI3K INHIBITOR PAXALISIB WITH NUCLEOSIDE ANALOG GEMCITABINE TO IMPROVE SURVIVAL OF ATYPICAL TERATOID/RHABDOID TUMORS

Abstract BACKGROUND Atypical teratoid/rhabdoid tumors (ATRT) have a dismal overall survival. Targeting the mTORC1/2 pathway kills ATRT tumor cells and increases the survival of mice bearing orthotopic xenografts. Paxalisib is a brain penetrant PI3K inhibitor that is in pediatric clinical trials for diffuse midline glioma. Paxalisib activates the integrated stress response (ISR) in ATRT. Consistent high-level activation of the ISR leads to cell death. We hypothesize paxalisib will synergize with the DNA-damaging agent gemcitabine, which also triggers the ISR, to over-activate this pathway and drive ATRT apoptosis. METHODS We analyzed ISR gene expression following mTOR inhibition via RNA-seq. Children’s Brain Tumor Network (CBTN) provided comparative data on ISR genes in ATRT and other pediatric brain tumors. Paxalisib/gemcitabine effects were evaluated through cell-based assays, western blot for ISR markers, and survival in three murine models. RESULTS ATRT have high baseline expression of ISR genes (CBTN: ATF4, eIF2α), and mTOR inhibition leads to activation of ISR genes (ATF4, CHOP, PPP1R15A, p<0.05). Paxalisib monotherapy significantly extends median survival in murine models compared to vehicle controls (CHLA-06: 40 to 54 days, p=0.001; BT-12: 21 to 35 days, p=0.05). Paxalisib/gemcitabine therapy synergized to slow cell growth and induce cell death (SynergyFinder BLISS score in five ATRT cell lines). Combination therapy leads to ISR activation (western blot: p-eIF2α, ATF4, CHOP). Combination slowed tumor growth (bioluminescent imaging) and led to significant extension of survival in orthotopic xenograft models (CHLA-06: 22 to 82.5 days; BT-37 56-day vehicle median survival while undefined combination survival). A third model is ongoing and showing similar results. CONCLUSIONS Paxalisib/gemcitabine therapy shows potential for targeting ATRT by over-activating the ISR. Our findings prompted the Pediatric Neuro-Oncology Consortium to consider this combination for inclusion in an upcoming clinical trial targeting relapsed/refractory ATRT, offering a novel approach to treat this aggressive tumor.

SiRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression

BackgroundBreast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets.MethodsTranscriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target.ResultsIntegrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours.ConclusionOur findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

Anti-PD-L1 therapy altered inflammation but not survival in a lethal murine hepatitis virus-1 pneumonia model.

Because prior immune checkpoint inhibitor (ICI) therapy in cancer patients presenting with COVID-19 may affect outcomes, we investigated the beta-coronavirus, murine hepatitis virus (MHV)-1, in a lethal pneumonia model in the absence (Study 1) or presence of prior programmed cell death ligand-1 (PD-L1) antibody (PD-L1mAb) treatment (Study 2). In Study 1, animals were inoculated intratracheally with MHV-1 or vehicle and evaluated at day 2, 5, and 10 after infection. In Study 2, uninfected or MHV-1-infected animals were pretreated intraperitoneally with control or PD-L1-blocking antibodies (PD-L1mAb) and evaluated at day 2 and 5 after infection. Each study examined survival, physiologic and histologic parameters, viral titers, lung immunophenotypes, and mediator production. Study 1 results recapitulated the pathogenesis of COVID-19 and revealed increased cell surface expression of checkpoint molecules (PD-L1, PD-1), higher expression of the immune activation marker angiotensin converting enzyme (ACE), but reduced detection of the MHV-1 receptor CD66a on immune cells in the lung, liver, and spleen. In addition to reduced detection of PD-L1 on all immune cells assayed, PD-L1 blockade was associated with increased cell surface expression of PD-1 and ACE, decreased cell surface detection of CD66a, and improved oxygen saturation despite reduced blood glucose levels and increased signs of tissue hypoxia. In the lung, PD-L1mAb promoted S100A9 but inhibited ACE2 production concomitantly with pAKT activation and reduced FOXO1 levels. PD-L1mAb promoted interferon-γ but inhibited IL-5 and granulocyte-macrophagecolony-stimulating factor (GM-CSF) production, contributing to reduced bronchoalveolar lavage levels of eosinophils and neutrophils. In the liver, PD-L1mAb increased viral clearance in association with increased macrophage and lymphocyte recruitment and liver injury. PD-L1mAb increased the production of virally induced mediators of injury, angiogenesis, and neuronal activity that may play role in COVID-19 and ICI-related neurotoxicity. PD-L1mAb did not affect survival in this murine model. In Study 1 and Study 2, ACE was upregulated and CD66a and ACE2 were downregulated by either MHV-1 or PD-L1mAb. CD66a is not only the MHV-1 receptor but also an identified immune checkpoint and a negative regulator of ACE. Crosstalk between CD66a and PD-L1 or ACE/ACE2 may provide insight into ICI therapies. These networks may also play role in the increased production of S100A9 and neurological mediators in response to MHV-1 and/or PD-L1mAb, which warrant further study. Overall, these findings support observational data suggesting that prior ICI treatment does not alter survival in patients presenting with COVID-19.

TMIC-03. TARGETING THE COLLAGEN RECEPTOR DDR1 PROMOTES ANTI-GLIOMA IMMUNITY BY REMODELING COLLAGEN FIBER ARCHITECTURE

Abstract Glioblastoma (GBM), characterized by its aggressive nature and profound immunosuppression, poses a significant therapeutic challenge with limited treatment options. We previously demonstrated that targeting Col1α1 eradicates oncostreams and improves survival in murine models. However, the underlying mechanisms by which collagen reshapes the glioma tumor microenvironment (TME) remain unknown. Through RNA-seq analysis we identified that DDR1, a collagen receptor, is overexpressed in genetically engineered glioma mouse models (GEMMs) including NPA (NRAS/shp53/shATRX), and NPD (NRAS/shp53/PDGFβ), compared to healthy brain tissue. Pharmacological inhibition of DDR1 enhanced radiosensitivity, disrupted oncostreams in vitro, and ex vivo, imaged with time-lapse confocal microscopy. GEMMs of DDR1 knockdown (NRAS/shp53/shATRX/shDDR1) using the Sleeping Beauty transposase system significantly increased median survival by over 50%. Gene Ontology analysis of differentially expressed genes between wild-type and DDR1 knockdown gliomas revealed enrichment in immune reactivity, cytokine-chemokine activity, and extracellular matrix. Inhibition of DDR1 enhanced intratumoral infiltration of CD45+, CD8+, and CD3+ immune cells, accompanied by a decrease in the abundance of immune-suppressive cells, suggesting that gliomas exploit DDR1-mediated mechanisms to promote immunosuppression and facilitate tumor growth. We hypothesize that DDR1 in glioma impedes immune surveillance by promoting collagen fiber alignment, as evaluated using collagen-specific second-harmonic generation microscopy. Furthermore, analysis of human datasets demonstrated a negative correlation between DDR1 expression and CD8, suggesting DDR1’s role as a key regulator of immune surveillance in gliomas. Interestingly, when DDR1 knockdown gliomas were implanted in immune deficient mouse models, NSG (NOD-scid-IL2Rγnull) or CD8−/− immune-deficient mice, no significant differences in survival were observed, indicating DDR1 expression in glioma cells dampens anti-glioma immunity. In conclusion, we propose that DDR1 within glioma cells induces an immune-inhibitory TME, reprograms cytokine and chemokine profiles, and promotes collagen fiber alignment, thus inhibiting anti-glioma immunity. Our data highlight DDR1 as a promising therapeutic target, providing a novel avenue for GBM treatment with significant potential.

Macropinocytosis As a Means of Selectively Targeting RAS-Mutant Multiple Myeloma

Introduction Roughly, 50% of multiple myeloma (MM) is driven by mutant RAS, both at presentation and more at relapse. Currently, in MM there are no clinical approaches to target this disease subset and ongoing strategies to inhibit RAS function or downstream effectors have had limited clinical success in other tumors. This has prompted our team to look into other more indirect modes of RAS-selective targeting. Oncogenic RAS-driven cancers use a unique actin-driven endocytosis pathway, termed macropinocytosis (MP), to increase the nutrient supply and sustain proliferation. To create a strategy for targeting RAS mutant MM, we have investigated whether RAS-mutant MM cells undergo MP and whether this process can be exploited as a therapeutic vulnerability, by utilizing this pro-oncogenic biochemical mechanism to selectively deliver a toxin to the MM clone using a MP selective monobody-drug conjugate (MDC). Methods The MDC relies on a proprietary fibronectin-based scaffold (monobody) identified based on its physiochemical properties that facilitates MP selective internalization, and therefore targets RAS-mutant MM tumor cells, with a very favorable pharmacokinetic profile. MMAE was attached to the monobody with a cathepsin-cleavable linker and a maleimide group for conjugation to the C-terminal free cysteine on the monobody protein (MDC-MMAE). MDC-MMAE was pre-clinically evaluated both in-vitro and in-vivo, in human MM cell lines, patient cells and a doxycycline inducible mutant KRAS cell system. Results In-vitro cell line and patient cell studies demonstrated that RAS-mutant MM cells undergo MP whereas wild type (WT) cells do not. To understand the role of mutant RAS in driving uptake of the MDC, a doxycycline-inducible mutant KRAS V12 KMS11 system was established. The functionality of the inducibility of the cell system was verified using protein expression and membrane ruffling in the presence of doxycycline. Fluorescently-labeled monobody (MDC-Cy5.5) and TMR-Dextran underwent a high level of uptake in the presence of induced mutant RAS. In contrast, in conditions where MP was absent, no uptake of MDC-Cy5.5 and TMR-Dextran was seen. Furthermore, when KMS11 KRASV12 and WT KMS11 were treated with MDC-MMAE, a 2-fold increase in cell death was observed in the mutant RAS cell line compared to wild type RAS. In-vivo studies showed that the pharmacological properties of the drug when conjugated to the monobody enabled faster serum clearance compared to antibody drug conjugates (ADCs) and nanomaterials, while maintaining cellular impermeability allowing enhanced drug uptake via MP. Importantly, in-vivo the MDC-MMAE technology had an improved toxicity profile compared to ADC-MMAE, increasing tolerable drug delivery by upwards of 15-fold. To test the anti-tumor effects in-vivo, the L363 (NRAS Q61), WT KMS11, and KMS11 KRASV12 cell lines were subcutaneously injected into MM murine models. After tumors were established, 0.5mg/kg MDC-MMAE was administered every 7 days. Mutant RAS L363 and KMS11 KRASV12 tumors treated with MDC-MMAE exhibited roughly a 6-fold decrease in tumor size after 21 days while WT KMS11 tumors showed no significant difference in tumor volume. MDC-MMAE at 5mg/kg showed complete tumor regression as a single agent in L363 tumors. Improved outcomes were seen with MDC-MMAE in combination with standard of care options at lower doses. Additionally, MDC-MMAE has displayed enhanced immunogenic cell death that has not been seen with other chemotherapeutic options, making it an ideal partner for immunotherapy combination. Conclusion Limited clinical options exist for the treatment of mutant RAS MM tumors. We show that by taking advantage of MP, a process upregulated in RAS-mutant cancers, MDC-MMAE specifically targets and inhibits MM growth and improves survival in murine models. It can also be combined with other MM standard of care drugs and with immunotherapy drugs to have a potentially important clinical effect. In collaboration with Tezcat Biosciences, the MDC-MMAE technology has achieved a phase II SBIR for advancement to clinical application.

Targeting Alpha-Ketoglutarate Disruption Overcomes Immunoevasion and Improves PD-1 Blockade Immunotherapy in Renal Cell Carcinoma.

The Warburg effect-related metabolic dysfunction of the tricarboxylic acid (TCA) cycle has emerged as a hallmark of various solid tumors, particularly renal cell carcinoma (RCC). RCC is characterized by high immune infiltration and thus recommended for immunotherapeutic interventions at an advanced stage in clinical guidelines. Nevertheless, limited benefits of immunotherapy have prompted investigations into underlying mechanisms, leading to the proposal of metabolic dysregulation-induced immunoevasion as a crucial contributor. In this study, a significant decrease is found in the abundance of alpha-ketoglutarate (αKG), a crucial intermediate metabolite in the TCA cycle, which is correlated with higher grades and a worse prognosis in clinical RCC samples. Elevated levels of αKG promote major histocompatibility complex-I (MHC-I) antigen processing and presentation, as well as the expression of β2-microglobulin (B2M). While αKG modulates broad-spectrum demethylation activities of histone, the transcriptional upregulation of B2M is dependent on the demethylation of H3K4me1 in its promoter region. Furthermore, the combination of αKG supplementation and PD-1 blockade leads to improved therapeutic efficacy and prolongs survival in murine models when compared to monotherapy. Overall, the findings elucidate the mechanisms of immune evasion in anti-tumor immunotherapies and suggest a potential combinatorial treatment strategy in RCC.

Targeting the Siglec-sialic acid axis promotes antitumor immune responses in preclinical models of glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain tumor, for which effective therapies are urgently needed. Cancer cells are capable of evading clearance by phagocytes such as microglia- and monocyte-derived cells through engaging tolerogenic programs. Here, we found that high expression of sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) correlates with reduced survival in patients with GBM. Using microglia- and monocyte-derived cell-specific knockouts of Siglec-E, the murine functional homolog of Siglec-9, together with single-cell RNA sequencing, we demonstrated that Siglec-E inhibits phagocytosis by these cells, thereby promoting immune evasion. Loss of Siglec-E on monocyte-derived cells further enhanced antigen cross-presentation and production of pro-inflammatory cytokines, which resulted in more efficient T cell priming. This bridging of innate and adaptive responses delayed tumor growth and resulted in prolonged survival in murine models of GBM. Furthermore, we showed the combinatorial activity of Siglec-E blockade and other immunotherapies demonstrating the potential for targeting Siglec-9 as a treatment for patients with GBM.

Effects of Gemcitabine/Nab-Paclitaxel and DMAPT in PDAC Cachexia in vivo and in vitro

Cachexia is the involuntary wasting of skeletal muscle and adipose tissue, affecting over 80% of patients with pancreatic ductal adenocarcinoma (PDAC). Gemcitabine and nab-paclitaxel (GemNP) combination are commonly given as first-line treatments for PDAC. Data from our lab showed GemNP reduced tumor growth and prevented muscle and fat wasting. Dimethylaminoparthenolide (DMAPT), a small molecule inhibitor, prevented muscle wasting and prolonged survival in a genetic murine model of breast cancer. It has yet to determine if GemNP in combination with DMAPT can improve indices of cachexia and overall survival. Therefore, I hypothesize that GemNP in combination with DMAPT will improve indices of cachexia and overall survival in a murine model of pancreatic cancer. Male 10-week-old C57BL/6J mice underwent orthotopic injection of 5x104 KPC cells or SHAM surgery. Mice were randomly assigned to 1 of 4 groups (N=10/group): SHAM+Vehicle, KPC+Vehicle, KPC+GemNP, KPC+GemNP+DMAPT. Gemcitabine (120mg/kg) and Nab-paclitaxel (10mg/kg) were injected intraperitoneally starting on day 4 and continued every 6 days. DMAPT (100mg/kg) was administered by gavage Monday-Friday. To determine the role of GemNP and DMAPT in vitro, KPC cells were treated with GemNP and/or DMAPT and cell viability evaluated. Additionally, we treated myotubes with GemNP with/without DMAPT to assess myotube diameter in an established KPC conditioned media. In KPC mice, GemNP (24.4days) increased survival compared to Vehicle (16.8days, p=.0007). However, the combination GemNP+DMAPT did not extend survival over GemNP alone (25.4days, p=.693). Tumor mass was similar between all groups (p=0.411). Interestingly, at the time of sacrifice, all KPC treated mice independent of treatment had similar reduction in adipose tissue and muscle mass compared to SHAM. In conclusion, the addition of DMAPT to GemNP did not extend survival over GemNP alone in an aggressive pancreatic tumor cell line. Future studies should determine if less aggressive tumor cell lines might benefit from GemNP and DMAPT.

IMMU-41. VERSATILE MRNA-NANOPARTICLE PLATFORM TO REPROGRAM THE BRAIN TUMOR MICROENVIRONMENT AND PREDICT TREATMENT OUTCOME

Abstract RNA vaccines have shown great promise as activators of immune responses against viral pathogens but their efficacy in cancer is unclear. Here, we describe a versatile, personalized mRNA-nanoparticle (RNA-NP) platform that can be customized to produce powerful anti-tumor immune activation, reprogram the brain tumor microenvironment, and predict vaccine efficacy just two days after treatment. Lipid mixtures (i.e. DOTAP, Cholesterol) with or without iron oxide nanoparticle-cores were complexed with mRNA encoding tumor antigens. Intravenous administration of RNA-NPs induced robust activation of innate immune cells resulting in prolonged survival in murine models of subcutaneous and intracranial melanoma. Inclusion of cholesterol in the lipid backbone enabled delivery of nucleic acids across the blood brain barrier and into tumor-associated myeloid cells in intracranial GL261 and KR158b tumors. These cholesterol-bearing liposomes not only activated innate immune cells in the tumor microenvironment (i.e. increased expression of CD80 and MHCII), but also enabled further manipulation of this compartment. Use of RNA-NPs to deliver siRNA targeting PD-L1 resulted in significant reduction in PD-L1 expression among tumor associated myeloid cells, leading to 37% long term survivorship in combination with systemic checkpoint blockade in an otherwise fatal model of GL261. We have also shown that IONPs incorporated into the cores of these particles enable non-invasive tracking of dendritic cells by MRI, enabling creation of a theranostic approach to: 1) enhance immunologic effects; 2) facilitate translocation across the blood-brain barrier; and 3) enable non-invasive imaging to predict response to RNA-NPs.

TMIC-13. GLIOMA-DRIVEN DENDRITIC CELL DYSFUNCTION AS AN IMMUNE EVASION STRATEGY AGAINST ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Our previous studies has developed an adoptive cellular therapy (ACT) against high grade gliomas in both human and murine systems. ACT significantly improves survival in murine models of CNS malignancies. Our studies have shown that efficacy is associated with the observed increase in tumor-associated dendritic cells (DC) which arise from transferred hematopoietic stem cells (HSC). HSC-derived DCs play a pivotal role in mounting the anti-tumor immune response. In this study, we have identified that treatment resistance may be due to glioma-driven dendritic cell dysfunction. We observed significant down regulation of co-stimulatory markers on tumor-associated dendritic cells in mice that escaped ACT and subsequent capacity to activate tumor-reactive T cells.Method: KR158B bearing mice received HSC and tumor reactive T cells one day before 9 Gy irradiation followed by 3 does of BMDC vaccine. Tumor associated DCs were sorted from tumors evaded from ACT and co-cultured with primary KR158B reactive T cells or escaped KR158B reactive T cells. T cell proliferation and interferon gamma were detected for comparison. PCR array was performed to identify dysregulated functional genes.Results and CONCLUSIONS Functional evidence demonstrated that dendritic cells from resistant tumor had significantly decreased capacity in activating tumor-reactive T cells against either primary KR158B glioma cells or T cells generated to target tumor cells that evaded ACT. Gene expression data showed this may be due to significant decreases in genes associated with co-stimulation, T cell engagement, and antigen presentation.

DDDR-28. EGFR AND SRC-MEDIATED ACTIVATION OF STAT3 DRIVES RESISTANCE TO MITOTIC INHIBITORS IN GLIOBLASTOMA, AND CAN BE REVERSED WITH FDA-APPROVED DRUGS

Abstract While the allure of targeted therapies in oncology has been their high degree of specificity and potency for key tumor drivers, they have been disappointing in glioblastoma (GBM), even for drugs that are blood brain barrier permeable and CNS retained. This point is highlighted by the experience with mitotic spindle inhibitors, drugs which block the G2M transition and induce mitotic catastrophe—a phenotype characterized by cell enlargement and polyploidy that leads to apoptotic cell death. We have shown that one of these, a potent inhibitor of the mitotic kinesin Kif11 (ispinesib), is highly active against GBM tumor initiating cells and prolongs survival in murine models of this disease. However, tumors eventually progress, reflecting the development of drug resistance. Although ispinesib resistant GBM cells develop mitotic catastrophe, they become highly resistant to the apoptosis that typically follows and continue to proliferate. We find that this apoptosis resistance requires phosphorylation of the transcription factor STAT3 at two residues—Y705 and S727. Phosphorylation of Y705, mediated by SRC kinase, translocates STAT3 to the nucleus where it induces transcription of anti-apoptotic proteins. Phosphorylation at S727, mediated by EGFR, translocates STAT3 to the mitochondria where it blocks release of cytochrome c—the penultimate effector in apoptosis. Simultaneously inhibiting both SRC and EGFR with FDA-approved, CNS permeant inhibitors reverses this resistance and significantly prolongs survival in ispinesib-treated GBM-bearing mice. Furthermore, we find that resistance to several other mitotic inhibitors also utilizes this STAT3-driven mechanism and can likewise be reversed with combined EGFR and SRC inhibition. Thus, our work demonstrates how a promising therapeutic approach, which has been disappointing in GBM, can in fact be rendered effective by anticipating and prospectively treating ab initio the mechanism that drives treatment resistance.

LOCL-03 INTRATUMORAL DELIVERY OF CONDITIONALLY REPLICATIVE HUMAN ADENOVIRUS 657 EXPRESSING THE IMMUNE CO-STIMULATOR CD40L (CRAD657-CD40L) AS A POTENTIALLY PROMISING TREATMENT FOR RECURRENT PEDIATRIC HIGH GRADE GLIOMA

INTRODUCTIONMalignancies of the central nervous system (CNS) have had largely unchanged survival outcomes despite decades of research. Recently, viral-based therapeutics have shown some benefit for patients with CNS malignancies in early clinical trials. Adenovirus has been demonstrated as safe and is currently being examined in several phase I and II clinical trials. We recently demonstrated that adenovirus expressing CD40L is effective in enhancing survival in murine models of diffuse midline glioma. Therefore, to enhance the tumor specificity of this virotherapy, we hypothesized that by using a novel conditionally replicative adenovirus expressing CD40L, CRAd657-CD40L, we would maintain this survival benefit in multiple murine models for high grade glioma while decreasing off-target toxicity. METHODSWe examined the utility of conditionally replicative adenovirus expressing CD40L in both in vitro and in vivo studies. Human cell lines from diffuse intrinsic pontine glioma (DIPG) and glioblastoma were used to confirm infectivity and CD40L expression, and syngeneic murine models of glioma were evaluated for toxicity and survival following intratumoral injection of a conditionally replicative adenoviral vector. RESULTSCRAd657-CD40L generated strong expression of CD40L in human in vitro DIPG XIII and U251 cell lines and induced MHCII expression on CD11c+ DC’s in U251/DC co-culture. Further, in syngeneic murine models of glioma, conditionally replicative adenoviral treatment significantly reduced toxicity while retaining survival efficacy. CONCLUSIONSGiven these promising results as well as the critical need for novel therapeutics in CNS malignancies, we are now progressing to human trials targeting pediatric HGG, an unmet need in pediatric neuro-oncology. This would be the first-in-human study using CRAd-657-CD40L in pediatric HGG. In this Phase 1 clinical trial, we hypothesize that intratumoral injection of CRAd657-CD40L will cause selective expression of CD40L, increased infiltration of immune cells into the tumor, and safely enhance tumor clearance.

P1197: AXICABTAGENE CILOLEUCEL (AXI-CEL) IN COMBINATION WITH RITUXIMAB FOR THE TREATMENT OF RELAPSED/REFRACTORY (R/R) LARGE B-CELL LYMPHOMA (LBCL): OUTCOMES OF THE PHASE 2 ZUMA-14 STUDY

Background: Despite the success of axi-cel, ≈60% of patients have no response or relapse within ~2 y after treatment (Jacobson C, et al. ASH 2021. #1764), highlighting the need for more therapeutic strategies. In preclinical studies, rituximab augmented CD19 chimeric antigen receptor (CAR) T-cell function and increased tumor reduction and survival in murine models via synergistic targeting with CAR T-cells (Mihara K, et al. Br J Haematol. 2010). Aims: Here, we report outcomes of ZUMA-14, a Phase 2, multicenter study of axi-cel in combination with rituximab in pts with R/R LBCL after ≥2 lines of systemic therapy. Methods: Eligible patients were ≥18 y with R/R LBCL. Patients received one rituximab dose (375 mg/m2) on Day -5, a conditioning regimen of cyclophosphamide and fludarabine on Days -5, -4, and -3, and a single axi-cel infusion of 2×106 CAR T cells/kg on Day 0. Starting on Day 21 post–axi-cel infusion, patients received 1 rituximab dose every 28 days for up to 5 doses. The primary endpoint was investigator-assessed complete response (CR) rate. Secondary endpoints included objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), safety, and biomarker assessments. The analysis reported here occurred after all treated patients had ≥12 months of follow-up. Results: As of 12/2/21, 27 patients were enrolled, and 26 received axi-cel and ≥1 rituximab dose (15 patients received all 6 rituximab doses); 1 patient discontinued treatment due to an adverse event (AE). Median age was 63 y (range, 38-82), 54% were male, 81% had stage III/IV disease, 62% had extranodal disease, 38% had elevated lactate dehydrogenase, and 85% had an age-adjusted International Prognostic Index (aaIPI) ≥1 (35% aaIPI=2). The CR rate was 65% (95% CI, 44%-83%), and the ORR was 88% (95% CI, 70%-98%). With a median follow-up of 17 months, 65% of the patients had ongoing response, with 57% ongoing in CR. Medians for DOR, PFS and OS were not reached. The estimated DOR and PFS rates at 12 months were 64% and 56%, respectively. The estimated 12-month OS rate was 76%, and 6 patients (23%) died of progressive disease. Most patients (92%) experienced Grade ≥3 AEs. Grade ≥3 cytopenias were reported in 85% of patients, with 38% ongoing on Day 30. Grade ≥3 neurologic events (NEs) occurred in 4 patients (15%); there was no Grade ≥3 cytokine release syndrome (CRS). Median times to onset of CRS and NEs were 4 days (range, 1-7 days) and 6 days (range, 3-32 days), respectively, with median durations of 5 days (range, 2-15 days) and 7 days (range, 1-39 days). No patients experienced myelodysplastic syndrome. Median peak CAR T-cell levels were comparable to the ZUMA-1 pharmacokinetic profile. Immune-modulating cytokines, including granzyme B, IL-6, CXCL10, IFN-γ and IL-2, were induced in patients following axi-cel and rituximab infusion and were more prominently elevated in responders vs non-responders. Peak rituximab levels were also elevated in responders vs non-responders. Summary/Conclusion: Results from ZUMA-14 demonstrated that axi-cel in combination with rituximab elicited a high CR rate and durable PFS with no new safety signals detected in patients with R/R LBCL.

Abstract 3033: Glucose deprivation promotes lung adenocarcinoma de-differentiation due to unbalanced EZH2 activity

Abstract Lung cancer is the most frequent cancer-related cause of death, and adenocarcinoma (LUAD) is the most frequent type. Despite the recent success of immunotherapies, survival of lung cancer patients has not significantly improved in the last decades. New therapies are necessary. We have previously identified sodium-glucose transporter 2 (SGLT2) as the major responsible for glucose uptake in LUAD, and we have showed that treatment with SGLT2 inhibitors significantly delays LUAD development and prolongs survival in murine models. However, our data shows that SGLT2 inhibitors also induce de-differentiation of LUAD cells, leading to a more aggressive phenotype and increased resistance to cisplatin. Glucose deprivation causes reduced αKG levels, leading to reduced activity of αKG-dependent histone demethylases and consequent histone hypermethylation. Supplementation of αKG or inhibition of the histone methyltransferase EZH2 reverse this phenotype, suggesting that this de-differentiated phenotype depends on insufficiency of αKG-dependent histone demethylases and unbalanced EZH2 activity. Consistently, double treatment with an SGLT2 inhibitor and an EZH2 inhibitor significantly reduces the tumor burden in a genetically engineered murine model of LUAD. We further characterized the effect of low glucose-induced tumor de-differentiation, identifying stabilization of hypoxia inducible factor 1α (HIF1α) as a major pathway responsible for the acquisition of a more aggressive phenotype following glucose deprivation. Finally, we identified an HIF1α-dependent transcriptional signature with prognostic significance in human LUAD. Our studies further our knowledge of the relationship between glucose metabolism and cell differentiation in cancer, characterizing the epigenetic adaptation of cancer cells to nutrient deprivation and identifying novel targets to prevent the development of resistance to metabolic therapies. Citation Format: Pasquale Saggese, Aparamita Pandey, Eileen Fung, Giorgio Giurato, Alessandro Weisz, Steven M. Dubinett, Claudio Scafoglio. Glucose deprivation promotes lung adenocarcinoma de-differentiation due to unbalanced EZH2 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3033.

Abstract 5536: Selinexor inhibits lymphoma-leukemia tumor progression directly and through activation of anti-cancer immune response

Abstract Background: Selinexor is a Selective Inhibitor of Nuclear Export (SINE) compound that binds covalently to Exportin 1 (XPO1) and inhibits its nuclear export function, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) including p53, pRB, and IκB-α. Selinexor was evaluated for improved survival in tumor-bearing mice with hematological malignancies using EL-4 (T cell lymphoma/leukemia) and C1498 (Acute myeloid leukemia) models. We hypothesized that selinexor increases survival by directly inducing tumor cell apoptosis and blocks immunosuppressive effects of myeloid derived suppressor cells (MDSCs). Methods: Mice were injected with 2×106 EL-4 or 1×106 C1498 cells intravenously followed by oral treatment with saline, vehicle, or different selinexor dosing schedules (5 mg/kg weekly (5), twice weekly on Wed and Fri (5 d1/3) or Mon and Fri (5 d1/5) or 10 mg/kg weekly (10). Selinexor was given by gavage. To examine the effect of selinexor on immune cells during tumor growth, Tumor bearing mice treated with vehicle or selinexor were euthanized at day 14 after tumor injection. Spleens were removed, single cell suspensions were isolated and the percentage of immune cells including MDSCs, regulatory T cells (Tregs) and the production of interferon gamma (IFN-γ) were analyzed by flow cytometry. Tumor bearing mice were monitored for survival in separate experiments. Results: Selinexor at the weekly 5 d1/3 and the 10 dosing significantly increased survival in mice injected with either tumor line. Whereas weekly 5 or 5 d1/5 dosing did not significant affect survival. Selinexor at weekly 10 dosing significantly reduced the population of polymorphonuclear MDSCs (PMN-MDSCs). Selinexor at 10, 5 d1/3 and 5/1/5 weekly dosing significantly increased IFN-γ production by CD8+ and CD4+ T cells when compared to vehicle. There were no differences in Tregs populations. Conclusions: These data demonstrate that different selinexor dosing and schedules have differential efficacy on survival in murine models of leukemia/lymphoma. Selinexor effects are dose and schedule dependent. We found that in contrast to the split dosing, the single weekly high dose of selinexor reduced splenic PMN-MDSC accumulation. While both dosing schedules increased the percentage of IFN-γ positive T cells in tumor bearing mice suggesting that selinexor’s anti-cancer activity is not limited only to direct cytotoxic or cytostatic tumor cells. These data support the concept that once weekly selinexor, at a higher dose (10 mg/kg (approximately 30 mg/m2) which is comparable to the clinical dose schedule (50-60 mg weekly) can modulate the immune system to target cancer cells and could be used alone or in combination with other immunotherapies to enhance the immune system. Citation Format: Hemn Mohammadpour, Yosef Landesman, Philip McCarthy. Selinexor inhibits lymphoma-leukemia tumor progression directly and through activation of anti-cancer immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5536.

Abstract 3825: Germline modifiers of the tumor immune microenvironment reveal drivers of immunotherapy response

Abstract With the continued promise of immunotherapy as an avenue for treating cancer, understanding how host genetics contributes to the tumor immune microenvironment (TIME) is essential to tailoring cancer risk screening and treatment strategies. Using genotypes from over 8,000 European individuals in The Cancer Genome Atlas and 137 heritable tumor immune phenotype components (IP components), we identified and investigated 532 TIME-SNPs. Focusing on 77 variants that were relevant to cancer risk, survival, or treatment response, we explored their potential to reveal novel targets for immunotherapy. Many variants overlapped regions with histone marks indicating active transcription, and influenced gene activities in specific immune cell subsets, such as macrophages and dendritic cells. TIME-SNPs implicated genes such as LAIR1, TREX1, CTSS, CTSW and LILRB2 were differentially expressed between responders and non-responders to immune-checkpoint blockade (ICB) in preclinical studies. Of these, LILRB2 and LAIR1 have already been identified as putative targets for immunotherapy. Here we found that inhibition of CTSS led to better tumor control and survival in murine models, alone or in combination with anti-PD-1. Collectively we show that through an integrative approach, it is possible to link host genetics to TIME characteristics, informing novel biomarkers for cancer risk and target identification in immunotherapy. Citation Format: Meghana Pagadala, Victoria Wu, Eva Pérez-Guijarro, Hyo Kim, Andrea Castro, James Talwar, Cristian Gonzalez-Colin, Steven Cao, Benjamin J. Schmiedel, Timothy Sears, Shervin Goudarzi, Divya Kirani, Rany M. Salem, Gerald P. Morris, Olivier Harismendy, Sandip P. Patel, Jill P. Mesirov, Maurizio Zanetti, Chi-Ping Day, Chun C. Fan, Wesley K. Thompson, Glenn Merlino, J. Silvio Gutkind, Pandurangan Vijayanand, Hannah Carter. Germline modifiers of the tumor immune microenvironment reveal drivers of immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3825.

Abstract 4235: PPRX-1701, a deliverable nanoparticle formulation of 6-bromo-acetoxime, blocks tumoral IDO1 expression and shows efficacy in immunocompetent murine glioblastoma models

Abstract Introduction: We previously showed that derivatives of the Chinese traditional medicine indirubin promote survival in murine models of the brain tumor glioblastoma. However, poor drug solubility hampered further development of this approach. Here we introduce PPRX-1701, a 6’-bromoindirubin acetoxime (BiA) containing drug/polymer nanoparticle formulation which can be injected intravenously at relatively high concentrations. Here we examine the efficacy and mechanism of PPRX-1701 in murine glioblastoma models. Methods: In vitro glioblastoma cell proliferation and migration assays were performed using standard approaches. Effects of PPRX-1701 on interferon-γ (IFN γ) signaling were determined by Western blotting. Delivery to murine GBM was assessed using a luminescent reporter assay, and survival studies were performed in immunocompetent murine glioblastoma models. Results: PPRX-1701 effects on glioblastoma cell proliferation and migration were indistinguishable from BiA alone. Our data show that intravenous administration of PPRX-1701 is well-tolerated and can reach intracranial murine glioblastoma as assessed by luminescent reporter assays. PPRX-1701 administration leads to improved survival in the GL261 glioblastoma mouse model (median survival 30 days (control), 47 days (treated), p < 0.0001). Treatment with PPRX-1701 was associated with alterations in the tumor immune microenvironment, with reduced Tregs and pro-tumor macrophages, and increased CD8+ T cells. Further mechanistic studies have shown that PPRX-1701/BiA blocks the expression of multiple immunosuppressive molecules in GBM downstream of interferon-γ (IFNγ) including PD-L1 and indoleamine 2,3-dioxygenase 1 (IDO1) - a key enzyme in the tryptophan-kynurenine-aryl hydrocarbon receptor (Trp-Kyn-AhR) immunosuppressive pathway. BiA promoted more effective T-cell mediated tumor cell killing using patient derived glioblastoma ex vivo co-culture models. Conclusion: This data supports further development of PPRX-1701 as a candidate immunotherapeutic agent for glioblastoma treatment. Ongoing pre-clinical studies are investigating combination with other relevant therapies. Citation Format: Mykola Zdioruk, Michal Nowicki, Bin Wu, Paul Boucher, Yuji Takeda, Weiyi Li, E Antonio Chiocca, Sean E. Lawler. PPRX-1701, a deliverable nanoparticle formulation of 6-bromo-acetoxime, blocks tumoral IDO1 expression and shows efficacy in immunocompetent murine glioblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4235.

Axicabtagene ciloleucel (axi-cel) in combination with rituximab (Rtx) for the treatment (Tx) of refractory large B-cell lymphoma (R-LBCL): Outcomes of the phase 2 ZUMA-14 study.

7567 Background: Despite the success of axi-cel, ≈60% of patients (pts) have no response or relapse within ̃2 y after Tx (Jacobson C, et al. ASH 2021. #1764), highlighting the need for more therapeutic strategies. In preclinical studies, Rtx augmented CD19 CAR T-cell function and increased tumor reduction and survival in murine models via synergistic targeting with CAR T-cells (Mihara K, et al. Br J Haematol. 2010). Here, we report outcomes of ZUMA-14, a Phase 2, multicenter study of axi-cel in combination with Rtx in pts with R-LBCL after ≥2 lines of systemic therapy. Methods: Eligible pts were ≥18 y with R/R LBCL. Pts received one Rtx dose (375 mg/m2) on Day -5, a conditioning regimen of cyclophosphamide and fludarabine on Days -5, -4, and -3, and a single axi-cel infusion of 2×106 CAR T cells/kg on Day 0. Starting on Day 21 post–axi-cel infusion, pts received 1 Rtx dose every 28 d for up to 5 doses. The primary endpoint was investigator-assessed complete response (CR) rate. Secondary endpoints included objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), safety, and biomarker assessments. The analysis reported here occurred after all treated pts had ≥12 mo of follow-up. Results: As of 12/2/21, 27 pts were enrolled, and 26 received axi-cel and ≥1 Rtx dose (15 pts received all 6 Rtx doses); 1 pt discontinued Tx due to an adverse event (AE). Median age was 63 y (range, 38-82), 54% of pts were male, 81% had stage III/IV disease, 62% had extranodal disease, 38% had elevated LDH, and 85% had an aaIPI ≥1 (35% aaIPI 2). The CR rate was 65% (95% CI, 44-83), and the ORR was 88% (95% CI, 70-98). With a median follow-up of 17 mo, 65% of the pts had ongoing response, with 57% ongoing in CR. Medians for DOR, PFS and OS were not reached. The estimated DOR and PFS rates at 12 mo were 64% and 56%, respectively. The estimated 12 mo OS rate was 76%, and 6 pts (23%) died of progressive disease. Most pts (92%) experienced Grade ≥3 AEs. Grade ≥3 cytopenias were reported in 85% of pts, with 38% ongoing on Day 30. Grade ≥3 neurologic events (NEs) occurred in 4 pts (15%), and there was no Grade ≥3 cytokine release syndrome (CRS). Median times to onset of CRS and NEs were 4 d (range, 1-7) and 6 d (range, 3-32), respectively, with median durations of 5 d (range, 2-15) and 7 d (range, 1-39). No pts experienced myelodysplastic syndrome. Median peak CAR T-cell levels were comparable to the ZUMA-1 pharmacokinetic profile. Immune-modulating cytokines, including granzyme B, IL-6, CXCL10, IFN-g and IL-2, were induced in pts following axi-cel and Rtx infusion and were more prominently elevated in responders vs non-responders. Peak Rtx levels were also elevated in responders vs non-responders. Conclusions: Results from ZUMA-14 demonstrated that axi-cel in combination with Rtx elicited a high CR rate with no new safety signals detected in pts with R-LBCL. Clinical trial information: NCT04002401.