Significant Prolongation Of Survival Research Articles

Abstract LB117: Virus-inspired Particles (ViPs) harnessing cytomegalovirus immune memory for local and systemic cancer immunotherapy

Abstract VerImmune is pioneering a First-in-Class treatment known as “Anti-tumor Immune Redirection” (AIR). This approach repurposes a patient’s pre-existing anti-viral or childhood vaccine immunity towards tumor cells for targeted destruction. VerImmune’s lead product, VERI-101, leverages pre-existing CD8+ T-cell immune memory acquired from past human cytomegalovirus (HCMV) infections. VERI-101 consists of VerImmune’s proprietary platform technology known as ViPs (Virus-inspired Particles) that are conjugated on their surface with a CD8+ T cell viral peptide antigen. ViPs are based on a modified mouse papillomavirus capsid protein of which 60 copies self-assemble into a 20-30nm T=1 icosahedral structure. In the case of VERI-101, the ViP is conjugated with an HLA-A*0201 restricted peptide epitope (NLVPMVATV, [NLV]), derived from the CMV pp65 antigen with an upstream furin protease cleavage site. The mechanism of action of AIR-ViPs including VERI-101 has been previously presented and involves three steps. Briefly, (1) VERI-101 targets the surface of the tumor cells and (2) enables the presentation of the CMV viral epitopes on their surface MHC, together (3) stimulating a recall of the pre-existing CMV memory response to attack the cancer cells (Dorrier et al., 2023 AACR). Here, we sought to study the in vivo anti-tumor efficacy of AIR-ViPs via both intra-tumoral and systemic treatment routes in two murine tumor models with pre-existing murine CMV (MCMV) immunity: (1) MC38 subcutaneous tumors for local and (2) B16.F10 lung metastasis for systemic treatments. As human CMV is species-specific, instead of VERI-101, a surrogate product, VERI-003 which redirects MCMV immunity, was tested as a single agent or in combination with anti-PD-1 in these tumor models. Our intra-tumoral studies with VERI-003 alone showed a dose-dependent anti-tumor effect (from 5 to 100ug) compared to no treatment controls. Surprisingly, no dose dependence was observed across the same three dose levels when combined with anti-PD-1, although significantly better anti-tumor effects occurred leading to complete tumor clearances in 50% of mice. Statistically significant prolonged survival was observed in most treated groups compared to controls. Our systemic studies with VERI-003 were consistent with our intratumoral studies in showing statistically significant single agent anti-tumor efficacy against B16.F10 lung metastases compared to no treatment. Anti-tumor effects of anti-PD-1 and combination were also observed. Additional studies are on-going to assess dose dependence and analysis of PK/PD effects. Taken together, our initial in vivo results demonstrate the tumor antigen-agnostic therapeutic potential of AIR and our AIR-ViP technology as a novel class of immuno-therapeutic drugs that could be used either as a monotherapy or in combination with checkpoint inhibitors. Citation Format: Cayce Dorrier, Felagot Abebe, Olivia Pak, Emily De-Bodene, Joshua Wang. Virus-inspired Particles (ViPs) harnessing cytomegalovirus immune memory for local and systemic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB117.

Co-targeting CD47 and VEGF elicited potent anti-tumor effects in gastric cancer

BackgroundCD47, serving as an intrinsic immune checkpoint, has demonstrated efficacy as an anti-tumor target in hematologic malignancies. Nevertheless, the clinical relevance of CD47 in gastric cancer and its potential as a therapeutic target remains unclear.MethodsThe expression of CD47 in clinical gastric cancer tissues was assessed using immunohistochemistry and Western blot. Patient-derived cells were obtained from gastric cancer tissues and co-cultured with macrophages derived from human peripheral blood mononuclear cells. Flow cytometry analyses were employed to evaluate the rate of phagocytosis. Humanized patient-derived xenografts (Hu-PDXs) models were established to assess the efficacy of anti-CD47 immunotherapy or the combination of anti-CD47 and anti-VEGF therapy in treating gastric cancer. The infiltrated immune cells in the xenograft were analyzed by immunohistochemistry.ResultsIn this study, we have substantiated the high expression of CD47 in gastric cancer tissues, establishing a strong association with unfavorable prognosis. Through the utilization of SIRPα-Fc to target CD47, we have effectively enhanced macrophage phagocytosis of PDCs in vitro and impeded the growth of Hu-PDXs. It is noteworthy that anti-CD47 immunotherapy has been observed to sustain tumor angiogenic vasculature, with a positive correlation between the expression of VEGF and CD47 in gastric cancer. Furthermore, the successful implementation of anti-angiogenic treatment has further augmented the anti-tumor efficacy of anti-CD47 therapy. In addition, the potent suppression of tumor growth, prevention of cancer recurrence after surgery, and significant prolongation of overall survival in Hu-PDX models can be achieved through the simultaneous targeting of CD47 and VEGF using the bispecific fusion protein SIRPα-VEGFR1 or by combining the two single-targeted agents.ConclusionsOur preclinical studies collectively offer substantiation that CD47 holds promise as a prospective target for gastric cancer, while also highlighting the potential of anti-angiogenic therapy to enhance tumor responsiveness to anti-CD47 immunotherapy.

Laser-Synthesized Germanium Nanoparticles as Biodegradable Material for Near-Infrared Photoacoustic Imaging and Cancer Phototherapy.

Biodegradable nanomaterials can significantly improve the safety profile of nanomedicine. Germanium nanoparticles (Ge NPs) with a safe biodegradation pathway are developed as efficient photothermal converters for biomedical applications. Ge NPs synthesized by femtosecond-laser ablation in liquids rapidly dissolve in physiological-like environment through the oxidation mechanism. The biodegradation of Ge nanoparticles is preserved in tumor cells in vitro and in normal tissues in mice with a half-life as short as 3.5 days. Biocompatibility of Ge NPs is confirmed in vivo by hematological, biochemical, and histological analyses. Strong optical absorption of Ge in the near-infrared spectral range enables photothermal treatment of engrafted tumors in vivo, following intravenous injection of Ge NPs. The photothermal therapy results in a 3.9-fold reduction of the EMT6/P adenocarcinoma tumor growth with significant prolongation of the mice survival. Excellent mass-extinction of Ge NPs (7.9 L g-1 cm-1 at 808nm) enables photoacoustic imaging of bones and tumors, following intravenous and intratumoral administrations of the nanomaterial. As such, strongly absorbing near-infrared-light biodegradable Ge nanomaterial holds promise for advanced theranostics.

Abstract 6660: The novel codon-modified Zika virus CDX-602 shows anti-tumor efficacy and remodels the tumor microenvironment in preclinical models

Abstract Multiple oncolytic viruses have been shown to induce multifaceted changes in the tumor microenvironment, that ultimately support induction of anti-tumor immune responses. We have previously demonstrated that CodaLytic, a codon-modified influenza virus, can kickstart the cancer immunity cycle at various steps in several mouse models with differing baseline immune contextures. Here, we are describing the activity of a new codon-modified virotherapeutic derived from the same synthetic viral engineering platform [Coleman et al., Science 2008]. Zika virus strain MR766 was synthetically codon-modified at 664 positions in the envelop gene to yield CDX-602. Virus was originally passaged in Vero cells and working stocks were expanded in BSR-T7 cells for in vivo use after titration on MA104.1 cells. Efficacy after intratumoral injection of 107 PFU/dose CDX-602 alone or in combination with 200 μg/dose αPD-1 blockade (RMP1-14, i.p.) was determined in subcutaneous B16-F10 melanoma and orthotopic EMT6 breast cancer models (n = 10). Changes in the tumor immune infiltrate were characterized using flow cytometry 6 or 8 days after treatment initiation, respectively (n = 5). In the immunotherapy-resistant B16-F10 model, CDX-602 monotherapy led to highly reproducible tumor growth inhibition (TGI, 52%, p < 0.0001 vs vehicle control), resulting in modest, but significant prolongation of median survival (25%, p < 0.01). Anti-tumor efficacy correlated with increased tumor immune infiltration with CD4+ and CD8+ T cells (R2 = 0.68 and 0.67, respectively, p < 0.01). Importantly, frequencies of cross-presenting dendritic cells (cDC) were increased 3.8-fold (p < 0.0001) after virus treatment, while no compensatory influx of CD206+ macrophages was observed, suggesting immune contexture changes conducive to anti-tumor immune induction. Efficacy was confirmed in the EMT6 model, in which monotherapy TGI was 44% and thus on par with αPD-1 blockade (50%, both p < 0.0001 vs control). Combination viroimmunotherapy in this model further improved efficacy, leading to 71% TGI and a doubling in complete regressions to 40%. Inclusion of CDX-602 in the treatment regimen led to similar changes in immune infiltration, in particular recruitment of CD8+ T cells and cDCs (9.7-fold and 6.9-fold increase after combination, respectively). Additional ex vivo analyses to confirm induction of anti-tumor immune responses are ongoing. Taken together, this preclinical data establishes CDX-602 as a novel virotherapeutic candidate that engages multiple mechanisms of action that contribute to anti-tumor activity. CDX-602 is the second virotherapeutic candidate derived from Codagenix’s codon modification platform, further supporting the utility of this synthetic viral engineering technology for viroimmunotherapy of cancer and highlighting its potential across multiple viral species. Citation Format: Nusrat Jahan, Yiwen Zhao, Katarina Blagovic, Ying Wang, J Robert Coleman, Steffen Mueller, Johanna K. Kaufmann. The novel codon-modified Zika virus CDX-602 shows anti-tumor efficacy and remodels the tumor microenvironment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6660.

Abstract 4107: Evaluating the efficacy of personalized neoantigen cancer vaccine in preclinical model: A promising step towards precision immunotherapy

Abstract Background: In the past decade, immunotherapy has transformed cancer treatment, with the development of immune-checkpoint inhibitors and adoptive cell therapies. A novel frontier is personalized vaccines targeting neoantigens unique to tumor cells. Vaccines based on neoantigens have several advantages. First, neoantigens are exclusively expressed by tumor cells and can, therefore, elicit truly tumor-specific T cell responses, thereby preventing ‘off-target’ damage to nonmalignant tissues. Second, neoantigens are de novo epitopes derived from somatic mutations, which presents the possibility to circumvent T cell central tolerance of self-epitopes and thus induce immune responses to tumors. One of the major challenges is the optimal neoantigen discovery/selection. Our study combines in silico HLA binding models with the 'MHC-PepSeq' platform to enhance neoantigen prediction, creating a personalized neoantigen peptide vaccine. We aim to demonstrate its efficacy in a mouse CT26 colon cancer model. Methods: Our method involved whole exome sequencing of tumor and normal DNA to identify neoantigens expressed in CT26 tumors, generating a peptide library. The peptides were further filtered using an in-silico prediction algorithm and our proprietary peptide-MHC binding assay (PepSeq assay), to determine those exhibiting the highest binding affinity with mice HLAs. Vaccines were prepared by dissolving these peptides in sterile PBS. Mice (BALB/C) were subcutaneously implanted with CT26 tumors, then immunized with the vaccine via IV injection in combination with a TLR7/8 agonist. Mice received a second vaccine dose on Day 14, and some groups were treated with an anti-PD-1 agent. Tumors and spleens from the mice were collected, processed into single-cell suspensions, and analyzed by single-cell sequencing, TCR analysis, IHC, and flow cytometry assays. Peripheral blood mononuclear cells (PBMCs) were collected and utilized in MHC tetramer assays. Results: Preliminary in vivo studies demonstrated significant tumor growth restrictions (TGI around 70%) and prolonged tumor-free survival in some mice. Our data showed the generation of neoantigen-specific T cells in vaccinated mice. Remarkably, vaccinated, tumor-free mice, when re-challenged with the same tumor cells, maintained their tumor-free status, suggesting the vaccine induced post-treatment immunological memory. Combining the vaccine with checkpoint inhibition resulted in better tumor growth restriction in mice (TGI around 93%). Conclusion: Overall, our data suggests that our personalized neoantigen vaccine platform has the potential to evoke robust and durable immune responses and, when paired with suitable complementary therapies and immune adjuvants, may serve as an effective cancer treatment. Citation Format: Tithi Ghosh Halder, Kate Gutowsky, Serina Ng, Trason Thode, Alexis Weston, Erin Kelley, Jorge Soria-Bustos, Jorge A. Giron, Taylor Bargenquast, Mohan Kaadige, Sanjana Tripuraneni, Brian Durbin, Shelby Rheinschmidt, Sydney Adamson, Michael Gordon, Justin Moser, John Altin, Raffaella Soldi, Sunil Sharma. Evaluating the efficacy of personalized neoantigen cancer vaccine in preclinical model: A promising step towards precision immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4107.

Abstract 1090: GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive pediatric solid tumor with poor survival and new targeted therapies are needed. Aims: Using an integrative proteogenomic quantitative approach, we sought to identify candidate immunotherapeutic targets then validate top targets in RMS preclinical models. Methods: We first performed plasma membrane enrichment followed by mass spectrometry (MS) to define the surfaceome of 7 fusion-positive (FP) and 14 fusion-negative (FN) RMS patient-derived xenograft models. Surface proteins were scored by IMMUNOTAR, our custom pipeline which extracts and integrates quantitative features from diverse proteogenomic and annotation datasets to provide a score for each protein, using mean-average-precision for score optimization. Cell surface expression of IMMUNOTAR-prioritized proteins was validated using flow cytometry, focusing on targets with immunotherapies in development. CAR T cells targeting the lead protein were engineered and studied in RMS preclinical models. Results: A total of 847 and 882 high confidence cell surface proteins were detected in FP and FN RMS surfaceome, respectively, with 94% of proteins overlapping the phenotypes. IMMUNOTAR ranked anaplastic lymphoma kinase (ALK), a putative immunotherapeutic target in FP RMS, as the top FP target. Glypican 2 (GPC2), a differentially expressed cell surface proteoglycan identified in neuroblastoma and other neural-derived tumors, targetable with CAR T cells and antibody-drug conjugates [Bosse et al. Cancer Cell 2017], ranked as the top protein in FN and 5th protein in FP RMS. Overall, GPC2 was abundantly expressed in 16 of 21 RMS models profiled. Flow cytometry revealed expression of GPC2 comparable to neuroblastoma in 5 of 8 RMS cells lines. Additionally, both FP and FN GPC2+ RMS cell lines (FN: RH36, SMS-CTR; FP: RH41, RH30) activated GPC2 CAR T cells with either a CD28 or a 4-1BB costimulatory domain, inducing release of IFN-gamma and IL-2 upon co-incubation across different E:T ratios as quantified by ELISA. Further, GPC2 CAR T cells induced potent RMS cell cytotoxicity compared to control CD19 CAR T cells with near 100% specific cytotoxicity at 1:1 E:T ratio. In a pilot study, GPC2 CAR T cells with 4-1BB costimulatory domains induced significant tumor regression (p=0.03) and prolonged progression-free survival (p=0.01) compared to CD19 CAR T cells in an established murine RH41 flank xenograft model. Conclusion: In summary, we probed the RMS surfaceome using MS and identified candidate proteins amenable for immunotherapeutic targeting using our IMMUNOTAR algorithm. We validated GPC2 as a candidate target in FP and FN RMS and are corroborating our findings in additional pre-clinical RMS models. Demonstration of GPC2 CAR efficacy in these models could lead to inclusion of RMS in our ongoing phase 1 clinical trial (NCT05650749) in a planned expansion phase. Citation Format: Rawan Shraim, Patrick M. Schuerch, Anna Maria Giudice, Karina L. Conkrite, Amber K. Weiner, Alexander B. Radaoui, Brian Mooney, Raushan T. Kurmasheva, Peter J. Houghton, Gregg B. Morin, Yael B. Mosse, Poul H. Sorensen, Benjamin A. Garcia, Ahmet Sacan, John M. Maris, Kristopher R. Bosse, Sharon J. Diskin. GPC2 directed CAR T cells are efficacious against fusion-positive and fusion-negative rhabdomyosarcomas [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 1090.

Abstract 6323: Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models

Abstract Background: CLL-1 is a compelling therapeutic target for AML as it is highly expressed on AML tumor cells and leukemic stem cells but is not expressed on hematopoietic stem cells. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology and leverages both checkpoint disruption and immune cloaking armoring strategies to potentially improve antitumor activity. The CB-012 anti-CLL-1 CAR was developed with a fully human scFv and the CD28 costimulatory domain and is currently in development for the treatment of relapsed or refractory AML (r/r AML). Here we describe preclinical studies that supported the CB-012 IND clearance by the FDA in October 2023. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012 through prevention of GvHD, PD-1 checkpoint disruption, and suppression of allograft rejection. In vitro and in vivo studies evaluated the specificity of antigen binding, antigen-dependent activity, and toxicologic potential. Results: CB-012 demonstrated potent antigen-dependent expansion and cytotoxic activity against CLL-1+ human AML cell lines and patient-derived cells in co-cultures. In AML xenograft models, a single dose of CB-012 CAR-T cells resulted in robust tumor control, leading to significant prolongation of survival. CB-012 co-culture with multiple CLL-1-negative cell types representing vital tissues demonstrated that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated highly specific interaction with human CLL-1, with no detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised murine models. Conclusion: CB-012, the first allogeneic anti-CLL-1 CAR-T cell therapy using both checkpoint disruption and immune cloaking armoring, demonstrated specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was demonstrated in an unbiased protein-binding study and no adverse safety signals were observed from CB-012 in murine toxicology models. These preclinical studies supported the IND clearance of CB-012, which is being evaluated in the AMpLify trial, a Phase 1, first-in-human clinical trial for patients with r/r AML (NCT06128044). Citation Format: Brian Francica, Elizabeth Garner, Sai Namburi, Cian Colgan, Tristan Fowler, Devin Mutha, Art Aviles, Morena Stanaway, Raymond Guo, Zili An, Erin Kelly, Emilie Degagne, George Kwong, Leslie Edwards, Emma Jakes, McKay Shaw, Benjamin Schilling, Jeremy Huynh, Ricky Luu, Max Sidorov, Rhonda Mousali, Mikk Otsmaa, Peter Lauer, Justin Skoble, Steven Kanner. Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6323.

Abstract 5470: ONC206 inhibits tumour growth and is a potential novel therapeutic strategy for refractory medulloblastoma

Abstract Medulloblastoma is the most common malignant pediatric brain tumor with a for certain molecular subgroups very low overall survival and therefore an urgent need for novel treatment approaches. Dordaviprone (ONC201) and its chemical derivative with nanomolar potency ONC206 are inhibitors of the dopamine receptor 2 (DRD2) and more importantly induce apoptosis of cancer cells by activation of the mitochondrial caseinolytic protease P (ClpP). ONC201 has shown objective responses in patients with diffuse midline gliomas and is currently being evaluated in the Phase 3 ACTION study. ONC206 is currently in Phase I clinical trials for pediatric and adult patients with primary brain tumors. In this study, we evaluated the preclinical therapeutic effects of ONC206 in medulloblastoma in vitro and in vivo and investigated the molecular mode of action for this imipridone. We found evidence for high expression of ClpP at both the RNA and protein level in medulloblastoma tumours, compared to very low expression in normal brain and normal cerebellum. In addition, we saw a pronounced reduction in cell viability of human Group 3 and Group 4 and murine medulloblastoma cells treated with ONC206 with extremely low IC-50s (range: 14nM - 2μM).After treatment with ONC206, we observed a significant downregulation of mitochondrial electron transport chain complexes I and III and mitochondrial hyperpolarization, as well as an induction of the ATF4 pathway, thereby implying that ONC206 induces an integrated stress response and mitochondrial damage. In order to test the efficacy of ONC206 in vivo, we used murine models of SHH-driven and Group 3 medulloblastoma as well as Group 3 patient-derived xenografts (PDXs). ONC206 led to a significant prolongation of survival in both murine models, with the SHH mice demonstrating a remarkable survival benefit. Mice from both Group 3 PDXs also responded to ONC206, with 25% of ONC206 treated animals exhibiting long-term tumor-free survival. Our results highlight ONC206 as a novel attractive therapeutic option for patients with relapsed medulloblastoma and importantly pave the way for a clinical trial testing the efficacy of ONC206 in the treatment of these patients. Citation Format: Theophilos Tzaridis, Jingbo Liu, Varun Prabhu, Robert Wechsler-Reya, Tobey MacDonald. ONC206 inhibits tumour growth and is a potential novel therapeutic strategy for refractory medulloblastoma [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 5470.

AXL inhibition suppresses early allograft monocyte-to-macrophage differentiation and prolongs allograft survival.

Innate immune cells are important in the initiation and potentiation of alloimmunity in transplantation. Immediately upon organ anastomosis and reperfusion, recipient monocytes enter the graft from circulation and differentiate to inflammatory macrophages to promote allograft inflammation. However, factors that drive their differentiation to inflammatory macrophages are not understood. Here, we show that the receptor tyrosine kinase AXL was a key driver of early intragraft differentiation of recipient infiltrating monocytes to inflammatory macrophages in the presence of allogeneic stimulation and cell-to-cell contact. In this context, the differentiated inflammatory macrophages were capable of efficient alloantigen presentation and allostimulation of T cells of the indirect pathway. Consequently, early and transient AXL inhibition with the pharmacological inhibitor bemcentinib resulted in a profound reduction of initial allograft inflammation and a significant prolongation of allograft survival in a murine heart transplant model. Our results support further investigation of AXL inhibition as part of an induction regimen for transplantation.

Clinical pharmacology profile of the claudin 18.2 antibody zolbetuximab.

316 Background: Zolbetuximab is an investigational first-in-class chimeric (mouse/human) monoclonal antibody directed against the tight junction protein claudin 18.2 (CLDN18.2). Zolbetuximab is currently being developed as first-line treatment of patients with locally advanced unresectable or metastatic HER2– gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors are CLDN18.2 positive, in combination with fluoropyrimidine- and platinum-containing chemotherapy. Methods: The clinical pharmacology of zolbetuximab (pharmacokinetics [PK] and impact of covariates, potential for drug-drug interactions and QTc prolongation, exposure-response [E-R] for efficacy and safety, immunogenicity [antidrug antibodies; ADAs]) was characterized from 9 studies in adults with advanced CLDN18.2-positive adenocarcinoma of the stomach, esophagus, or GEJ. Results: Zolbetuximab exhibited dose-proportional PK at doses of 33 to 1000 mg/m2. Based on population PK modeling using data from 714 participants, zolbetuximab exposure (Table) was not meaningfully affected by sex, race/ethnicity, age, mild/moderate renal impairment, or mild hepatic impairment. Gastrectomy was predicted to increase Cave by 36%. Coadministration of zolbetuximab with chemotherapy did not show clinically meaningful changes in drug exposures of zolbetuximab or chemotherapy. At therapeutic doses, zolbetuximab had no clinically meaningful effect on QTc prolongation. In phase 3 studies, zolbetuximab (800 mg/m2 loading dose/600 mg/m2 Q3W) had a manageable safety profile and was associated with significant prolongation of progression-free survival (PFS) and overall survival (OS). E-R analyses suggested that higher zolbetuximab exposure may further prolong PFS and OS but with increased probability of gastrointestinal events and infusion-related reactions. E-R model simulations suggested comparable zolbetuximab efficacy and safety between the 800/600 mg/m2 Q3W regimen and an alternative 800/400 mg/m2 Q2W regimen for use in combination with chemotherapy. ADAs were detected in 4.4% of patients in phase 3 studies with no apparent impact on zolbetuximab PK, efficacy, or safety. Conclusions: The clinical pharmacology of zolbetuximab was well characterized using data from 9 clinical trials. The integrated data support the proposed 800/600 mg/m2 Q3W regimen as well as an 800/400 mg/m2 Q2W regimen in combination with chemotherapy. Clinical trial information: NCT00909025 , NCT01671774 , NCT03528629 , NCT04086758 , NCT01197885 , NCT01630083 , NCT03505320 , NCT03504397 , NCT03653507 . [Table: see text]

Differential Distribution of the DNA-PKcs Inhibitor Peposertib Selectively Radiosensitizes Patient-derived Melanoma Brain Metastasis Xenografts.

Radioresistance of melanoma brain metastases limits the clinical utility of conventionally fractionated brain radiation in this disease, and strategies to improve radiation response could have significant clinical impact. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is critical for repair of radiation-induced DNA damage, and inhibitors of this kinase can have potent effects on radiation sensitivity. In this study, the radiosensitizing effects of the DNA-PKcs inhibitor peposertib were evaluated in patient-derived xenografts of melanoma brain metastases (M12, M15, M27). In clonogenic survival assays, peposertib augmented radiation-induced killing of M12 cells at concentrations ≥100 nmol/L, and a minimum of 16 hours exposure allowed maximal sensitization. This information was integrated with pharmacokinetic modeling to define an optimal dosing regimen for peposertib of 125 mpk dosed just prior to and 7 hours after irradiation. Using this drug dosing regimen in combination with 2.5 Gy × 5 fractions of radiation, significant prolongation in median survival was observed in M12-eGFP (104%; P = 0.0015) and M15 (50%; P = 0.03), while more limited effects were seen in M27 (16%, P = 0.04). These data support the concept of developing peposertib as a radiosensitizer for brain metastases and provide a paradigm for integrating in vitro and pharmacokinetic data to define an optimal radiosensitizing regimen for potent DNA repair inhibitors.

Endometrial cancer PDX-derived organoids (PDXOs) and PDXs with FGFR2c isoform expression are sensitive to FGFR inhibition

Endometrial cancer (EC) patients with metastatic/recurrent disease have limited treatment options and poor survival outcomes. Recently, we discovered the FGFR2c splice isoform is associated with poor prognosis in EC patients. Here we report the establishment of 16 EC patient-derived xenografts (PDX)-derived organoids (PDXOs) with or without FGFR2c expression. In vitro treatment of 5 EC PDXOs with BGJ398 showed significant cell death in 3 models with FGFR2c expression. PDXs with high/moderate FGFR2c expression showed significant tumour growth inhibition (TGI) following 21-day treatment with FGFR inhibitors (BGJ398 or pemigatinib) and significantly prolonged survival in 4/5 models. Pemigatinib + cisplatin combination therapy (n = 5) resulted in significant TGI and prolonged survival in one of two p53abn PDXs. All five models treated with cisplatin alone showed de novo resistance and no survival benefit. Seven-day treatment with BGJ398 revealed a significant reduction in angiogenesis and CD206 + M2 macrophages. These data collectively support the evaluation of FGFR inhibitors in a clinical trial.

Hijacking of the Fibrinolytic System By B-Cell Acute Lymphoblastic Leukaemia and Its Therapeutic Targeting

The fibrinolytic system plays a role in the mobilization of hematopoietic stem cells from their bone marrow microenvironment (BMM), but its function in leukemias is not well understood. The fibrinolytic system consists of the proenzyme plasminogen and its active form, plasmin, a serine protease. The activation of plasmin is tightly regulated by activators such as tissue plasminogen activator (tPA) and various inhibitors. A role of this plasminogen activation system in the microenvironment of solid tumors is being unravelled, but a function of this pathway in the BMM, where hematological cancers usually originate, has so far been elusive. Transcriptome analysis of mesenchymal cells in the BMM of mice with different leukemias had revealed annexin A2 ( Anxa2), an initiator of fibrinolysis, to be differentially expressed. Using an ANXA2-deficient BMM as a model for impaired fibrinolysis, we showed that ANXA2 promotes the activation of plasmin, leading to decreased levels of the extracellular matrix (ECM) protein fibronectin in the BMM and accelerating the progression of BCR-ABL1 + B-cell acute lymphoblastic (B-ALL), but not MLL-AF9 + acute myeloid leukemia. Consistently, induction of B-ALL in tPA-deficient mice led to fibronectin accumulation in the BMM and significant survival prolongation. The dense ECM in an ANXA2-deficient BMM is shown to entrap insulin-like growth factor (IGF) and to specifically reduce mTORC2-dependent signalling and proliferation in B-ALL cells. Conversely, B-ALL cell-derived interleukin (IL)-6 conditions hepatic generation of plasminogen, the precursor of plasmin, perpetuating this circuit. Treatment with -aminocaproic acid (EACA), an anti-hemorrhagic drug, which inhibits plasmin activation, reduces tumor burden and prolongs survival in syngeneic and xenogeneic murine models via increased fibronectin in the BMM. Human data confirm that IGF1 and fibronectin staining in trephine biopsies are correlated and that the mTORC2 pathway is distinctly activated in B-ALL. Higher expression of SERPINF2 (α2-antiplasmin), an inhibitor of plasmin, in human B-ALL cells leads to superior outcome. In summary, B-ALL-derived IL-6 impacts hepatic generation of fibrinolytic agents influencing B-ALL progression via ECM remodeling and regulation of the availability of B-ALL-supportive growth factors. Inhibition of plasmin-mediated degradation of the ECM by EACA may be beneficial as adjunct therapy in B-ALL.

The Potent Dihydroorotate Dehydrogenase Inhibitor, Hosu-53, Exhibits Compelling Monotherapy Efficacy in Multiple Myeloma and Augments CD47 Targeted Therapy

Worldwide, multiple myeloma (MM) is the second most common hematological malignancy characterized by the expansion of aberrant mature plasma cells in the bone marrow. Despite the FDA approval of several drugs with different mechanisms of action such as immunomodulatory drugs (IMiDs) and proteosome inhibitors (PIs), MM remains a challenging incurable disease where most patients ultimately relapse and become refractory to available . The resistant nature of MM underscores the need for novel therapeutic strategies that can directly affect MM cells while enhancing the outcome with MM current standard of care (SOC). Among the most promising MM SOC agents is monoclonal antibodies (mAb) such as the CD38 antibody daratumumab, which significantly improved the management of newly diagnosed and relapsed/refractory MM. Therefore, we aimed to develop novel therapies that can enhance mAb therapy in MM. Dihydroorotate dehydrogenase (DHODH) is an enzyme which mediates the fourth and rate-limiting step in the de novo pyrimidine synthesis pathway converting dihydroorotate to orotate, the precursor of uridine. Pyrimidine starvation using DHODH inhibitors has been shown to have potent antiproliferative activity in several malignancies including acute myeloid leukemia (AML) and MM. Thus, we developed a novel potent DHODH inhibitor, HOSU-53 for the treatment of hematological malignancies. We previously discovered the ability of HOSU-53 to modulate surface CD38 expression and found that HOSU-53 had potent monotherapy activity in MM and provided impressive synergy in combination with daratumumab using the NCI-H929 MM cell line derived xenograft (CDX) model (2022 AACR annual meeting abstracts). Herein, we further expanded our studies to pre-clinically develop HOSU-53 as a new potential MM therapy and explore additional synergistic immunotherapy combinations that could maintain efficacy in the case of anti-CD38 therapy resistance. We evaluated the in vitro antiproliferative efficacy of HOSU-53 against a panel of MM cell lines and found nanomolar potency validating that HOSU-53 would have monotherapy efficacy. Indeed, we conducted in vivo studies using two additional MM CDX subcutaneous (s.c) models, OPM-2 and RPMI-8226, and found significant tumor delay and survival advantage. In the OPM-2 model, HOSU-53 has a median survival of 54-days compared to vehicle at 28-days, while the median survival for HOSU-53 was 60-days compared to vehicle at 26-days in the RPMI-8226 model. We further verified HOSU-53 efficacy using the disseminated MM1.S luciferase CDX model and found a significant prolonged survival in the HOSU-53 cohort (median survival53-days) compared to vehicle (median survival28-days) that was further enhanced with isatuximab combination resulting in superior survival benefit (median survival 69-days). Given the continuous challenge in maintaining a durable response with anti-CD38 therapies due to resistance, we sought to explore additional combination regimens to maximize HOSU-53 efficacy. Currently there is significant clinical interest in CD47 antibody therapy such as magrolimab for both solid tumors and hematological malignancies. Our previous work suggested a strong synergy between CD47 antibodies and HOSU-53 in AML with curative potential (2022 ASH annual meeting abstracts), and hypothesized that we would observe similar synergy with HOSU-53 in combination with anti-CD47 in MM. Indeed, we found complete tumor regression in all mice treated with HOSU-53 + B6.H12 anti-CD47 therapy in the NCI-H929 s.c CDX model and significant prolonged survival and reduced bioluminescence in the NCI-H929 luciferase disseminated CDX model. Together, these two studies validate a potential clinical benefit to explore HOSU-53 + anti-CD47 regimen in MM patients. Furthermore, we observed that calreticulin (pro-phagocytosis signal) was modulated post HOSU-53 in vitro treatment suggesting its role in the observed synergy between HOSU-53 and CD47 blockade therapy. In summary, we show compelling survival benefit for HOSU-53 as a monotherapy which is further enhanced when combined with anti-CD38 or anti-CD47 therapies. HOSU-53 is expected to enter phase 1 clinical trials in 2024 and our data is supportive for its expansion into MM.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p<0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p<0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

CLL-1-Targeted Allogeneic CAR-T Cells Exhibit High on-Target Specificity and Potent Cytotoxicity in Preclinical Models of Acute Myeloid Leukemia

Background: CLL-1 is a compelling therapeutic target for acute myeloid leukemia (AML) as it is highly expressed on AML tumor cells and leukemic stem cells, but is not expressed on hematopoietic stem cells. An allogeneic anti-CLL-1 CAR-T cell therapy (CB-012) is in development for relapsed or refractory (r/r) AML. The CAR was generated with a fully human scFv targeting CLL-1 that was selected from a panel of scFvs. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology to leverage both checkpoint disruption and immune cloaking for potentially improved antitumor activity. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to reduce graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding to prolong antitumor activity. This multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012. In vitro and in vivo studies evaluated specificity for antigen binding, antigen-dependent activity, and preclinical safety assessments. Results: CB-012 CAR-T cells express a fully human anti-CLL-1 scFv-containing CAR construct and demonstrate potent antigen-dependent cytotoxic activity in human AML cell line co-cultures. In AML xenograft models, a single-dose administration of CB-012 CAR-T cells exerted robust tumor control, leading to significant prolongation of survival. Cell binding studies suggested that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity when examined in the context of a collection of cell types representing vital tissues. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated specific interaction with human CLL-1, without detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised mouse models. Conclusion: CB-012 demonstrated highly specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was further demonstrated by screening in an unbiased protein-binding study and no adverse safety signals were observed in murine models. These data support advancing the development of CB-012 into a first-in-human clinical trial for patients with r/r AML.

Targeting Epigenetic Resistance Mechanisms to PI3 Kinase Inhibition in Leukemic Stem Cells

Acute myeloid leukemia (AML) is a heterogeneous disease associated with poor survival and frequent relapse rates due to the inability to effectively target leukemic stem cells (LSCs) and acquired resistance to existing treatments. Development of treatments targeting LSCs and overcoming acquired resistance is crucial to improve the survival of AML patients. The PI3 kinase (PI3K)/AKT signaling pathway is activated in 80% of AML patients, across various AML subtypes, making PI3K an attractive therapeutic target for this disease. Several PI3K inhibitors, such as copanlisib, are clinically approved for other indications, with a reasonable toxicity profile. The effects of PI3K inhibitors on LSCs however, remains unknown. Several catalytic isoforms of PI3K are expressed in hematopoietic cells. To determine whether these PI3K isoforms play a role in LSC self-renewal, we performed colony assays on KMT2A-AF9 retrovirally transduced hematopoietic stem and progenitor cells (HSPCs) with either genetic deletion or pharmacologic inhibition of PI3K isoforms. We found that disruption of PI3K signaling reduces the self-renewal of LSCs and promotes myeloid differentiation in both mouse LSCs and human AML cell lines. KMT2A-AF9 induces leukemic transformation via aberrant upregulation of key self-renewal genes in hematopoietic progenitors, such as HoxA9 and Meis1 (PMID: 16862118). We found that pharmacologic inhibition of PI3K isoforms leads to a reduction in HoxA9 and Meis1 expression in LSC colonies. In the KMT2A-AF9 retroviral bone marrow transplantation model of AML, genetic deletion of the PI3K isoform Pik3ca leads to a reduction in LSC numbers in the bone marrow and prolonged survival (Fig 1A), although it is not curative. In addition, we observed significant prolongation of survival upon secondary transplantation of Pik3ca leukemic cells, suggesting a reduction in functional LSCs. Transcriptome analysis on LSCs isolated from pre-clinical bone marrow aspirates of KMT2A-AF9 primary transplant mice revealed downregulation of the target genes of HoxA9, an important driver of LSC self-renewal. We also observed enrichment of the targets of EZH2, a histone methyltransferase that catalyzes the H3K27 trimethylation repressive mark. AKT is known to phosphorylate EZH2 at Ser21, leading to inhibition of EZH2 function (PMID: 16224021). Therefore, we hypothesized that Pik3ca deletion can promote LSC differentiation by de-repressing EZH2 activation via AKT inactivation. To determine whether PI3K inactivation differentiates KMT2A-AF9 leukemic cells via dephosphorylation of EZH2, we performed Western blot analysis on bone marrow cells from Pik3ca KO and WT leukemic mice. As expected, Pik3ca deletion significantly reduced AKT phosphorylation. Unexpectedly, a reduction of total EZH2 protein levels was also observed in Pik3ca KO leukemic cells (Fig 1B), suggesting that EZH2 downregulation may be a non-genetic acquired resistance mechanism to PI3K inactivation. Histone proteomics analysis at two different timepoints after treatment with the PI3K inhibitor copanlisib revealed rapid and dynamic changes in histone modifications, including H3K27me3 and H3K4me3, consistent with epigenetic mechanisms of resistance to PI3K inhibition. EZH1, a homolog of EZH2, has been reported to compensate for EZH2 function (PMID: 26219303, PMID: 31189531). Thus, we hypothesize that PI3K inhibition may sensitize leukemic cells to EZH1/2 dual inhibition by reducing EZH2 levels. We found that dual inhibition with copanlisib and the EZH1/2 inhibitor valemetostat leads to a combinatorial effect to strongly decrease the proliferation of AML cell lines and AML patient samples. Additionally, we found that treatment of KMT2A-AF9 AML mice with these compounds induces myeloid differentiation and leads to a reduction in leukemia initiating cell capacity, as demonstrated by prolonged survival upon secondary transplantation. Overall, our data suggests that PI3K plays an important role in leukemic stem cells, and that EZH2 downregulation is a non-genetic mechanism of resistance to PI3K inhibition. Additionally, our findings indicate that combining PI3K inhibitors with EZH1/2 dual inhibitors could be a promising therapeutic approach to reduce LSCs, prevent resistance, and prolong survival in AML patients.

EXTH-65. IDENTIFICATION OF LOMERIZINE AS A POTENTIAL ANTI-GLIOBLASTOMA DRUG

Abstract BACKGROUND Glioblastoma (GBM) is one of the most malignant primary brain tumors. Despite development of surgical techniques, chemotherapy and radiotherapy, GBM prognosis is still poor. Here, we identified lomerizine, a prophylactic drug for migraine, as a potential anti-GBM drug from thousands of existing compounds. We investigated its therapeutic effects both in vitro and in vivo with the purpose of drug repositioning. Materials and method: Human patient-derived glioma stem cell line (GSC; KGS01, KGS10, KGS15), their differentiated cells, and GBM cell lines (A172, SNB19, T98, U87) were used. Proliferation assay, migration assay and invasion assay were performed with all cell lines. Sphere-forming assay was performed with GSCs. Various signaling pathways altered by lomerizine were analyzed by Western blotting. In addition, induction of apoptosis was evaluated by immunofluorescence and Annexin V assay. Anti-GBM effects of lomerizine in vivo was validated with a mouse brain tumor model. Result: Lomerizine inhibited proliferation, migration, and invasion dose-dependently in all cell lines, especially in GSCs. Sphere formation was also suppressed by lomerizine in all GSCs. STAT3, which is involved in tumorigenesis, was dephosphorylated after the treatment with lomerizine in a dose-dependent manner in all cell lines. Furthermore, apoptosis induction by lomerizine was confirmed in both immunofluorescence and Annexin V assay. In vivo experiments showed a significant tumor suppression and prolongation of overall survival in the group of mice treated with lomerizine. CONCLUSION This study suggests that lomerizine has anti- GBM effects.

Abstract 376: Comparison of Pre and Post Conditioning With Poloxamer 188 in Rat Model of Severe Hemorrhagic Shock

Background: Hemorrhagic shock remains a leading cause of morbidity and mortality after trauma. Poloxamer (P) 188, an FDA-approved copolymer-based cell membrane stabilizer, is a nonionic triblock copolymer believed to prevent cellular injury after ischemia and reperfusion. While post conditioning may be used in a broad range of scenarios, pre-conditioning may add improved efficacy in scenarios where hemorrhage can be expected, the peri-operative period. We have previously demonstrated prolonged survival with P188 post-conditioning however without complete rescue, here we hypothesize pre-conditioning would further improve survival. Methods: 35 male Sprague Dawley rats (564+/-73 g) were anesthetized, ventilated and instrumented. All rats underwent rapid controlled blood loss of 40% estimated blood volume over 30 min, followed by prolonged permissive hypotension for 60 min before re-transfusion of the lost blood to euvolemia and up to 6 hrs of monitoring under general anesthesia. Rats were then randomized to 3 groups: 10 rats received a 0.8 cc iv bolus of 250mg/kg P188 15min before hemorrhage (pre), 12 rats received the same dose of P188 after hemorrhage (post), and 13 control rats only received 0.8 cc vehicle. Primary endpoint was survival time from the start of hemorrhage. Data are median and IQ range; statistics: Kruskal-Wallis and Chi-Square tests, *p<0.05. Results: P188 administration immediately after hemorrhage significantly improved median survival time compared to both the pre and control groups [post *245 (49, 314) min vs. control group; 53 (32-60), vs pre; 59 (24-70) min, p = 0.0174, 0.00045, respectively]. However, there was no significant differences in survival time between pre and control grouyps and there were no differences in survival rate at 6 hours (pre; 0% vs post; 8% vs control; 7%). Conclusion: A bolus of P188 given immediately after a severe hemorrhagic insult prolonged survival time significantly. However, administration of P188 15 min before hemorrhage did not produce a significant prolongation of survival time.P188 is a highly promising compound whose dose, application timing and application duration in severe hemorrhagic shock require more research.

TRANSLATIONAL RESEARCH-03 MULTIPLE MECHANISMS MODULATE TEMOZOLOMIDE RESISTANCE AND ARE POTENTIAL TARGETS FOR GLIOBLASTOMA THERAPY.

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Temozolomide (TMZ) is the only drug currently been used for treatment of newly diagnosed GBM patients but the benefit from this agent has been hampered by the primary and secondary resistance. Thus, there is a strong need for uncovering the mechanisms of TMZ resistance. The DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) is the main driver of primary TMZ resistance and lacking MGMT expression due to promoter hypermethylation has been associated with a significant survival prolongation. However, the MGMT-independent resistance mechanisms remain poorly understood. To investigate TMZ resistance mechanisms, we developed in vivo TMZ resistant models using GBM patient derived xenografts (PDXs) by treating animals bearing flank PDXs with clinically relevant 3-cyles of TMZ. The recurrent tumors were all resistant to TMZ through multiple mechanisms, including MGMT re-expression, elevated homologous recombination (HR) and/or non-homologous end joining (NHEJ) repair activities, and loss of mismatch repair (MMR). However, other tumors developed resistance due to unknown mechanisms. To identify novel modulators of TMZ sensitivity, we performed genome wide screening using shRNA library, and we identified over 600 druggable targets. Validation experiments identified RBBP4 gene as one of the candidates involved in TMZ sensitivity. Silencing RBBP4 in GBM cells sensitized both MGMT-methylated and -unmethylated GBM cells to TMZ. Mechanistically, RBBP4 interacts with p300 to form a chromatin remodeling complex that control expression of DNA damage repair genes, and silencing RBBP4 or p300 significantly blocked the recovery of TMZ induced DNA double strand breaks causing cell death. We also found the nuclear pore exporter XPO1 is the modulator of TMZ sensitivity, and the XPO1 inhibitor Selinexor is currently being evaluated for GBM treatment in a clinical trial. Collectively, our work has uncovered multiple mechanisms of TMZ resistance that are potential targets for GBM therapy.