Immunotherapy against Gliomas

Immunotherapy has been demonstrably effective against various cancers, particularly those in the hematopoietic system and those with a high tumor-specific antigenic burden. Unfortunately, the development of immunotherapeutic strategies has proven more challenging against central nervous system (CNS) malignancies due to several unique characteristics of brain tumors that pose extraordinary barriers. To date, there is a lack of phase III trials demonstrating improved progression-free survival (PFS) and/or overall survival (OS) using immunotherapies in brain cancers. However, a better mechanistic understanding of current resistance to immunotherapies along with data from novel innovative techniques to overcome these barriers has been encouraging. This chapter gives an overview of current immunotherapies in the development of brain cancers. We will evaluate the present studies available in the clinical setting and any of their potential findings. The chapter will also discuss pertinent preclinical strategies whose translation for human use would potentially prove efficacious or provide invaluable scientific discovery.

Dnmt3a Mutations in the Hematopoietic System Promote Colitis-Associated Colon Cancer: A Model of Clonal Hematopoiesis in Solid Tumors

Clonal hematopoiesis (CH) is a condition wherein hematopoietic stem cells (HSCs) acquire mutations, often in presumed leukemia driver genes such as in DNMT3A, leading to clonal dominance and disproportionate contribution to peripheral blood without overt hematologic abnormalities (Jaiswal et al. 2014). In large cohorts of patients with solid tumors including colorectal cancer, presence of CH is associated with inferior overall survival due to progression of primary malignancy (Coombs et al. 2017). While potential involvement of CH in the pathogenesis of solid tumors driven by unrelated genetic alterations has far-reaching translational implications, the mechanistic understanding of this relationship is lacking. Colorectal cancer is a leading cause of cancer deaths worldwide and can arise from preceding inflammatory bowel disease. More than 20% of patients with colon cancer have detectable CH, which is notably more prevalent than in age-matched general population. We hypothesized that bone marrow-derived cells harboring alterations in DNMT3A, the most commonly disrupted gene in CH (up to 40% of cases), contribute to the pathogenesis of colorectal cancer. To address this, we combined a bone marrow transplantation-based model of CH driven by heterozygous loss of Dnmt3a with a well-established induction of colitis-associated colon cancer (CAC) by azoxymethane (AOM, a carcinogen) and dextran sodium sulfate (DSS, a colitogen). High resolution colonoscopy demonstrated increased colon wall opacity, visible bleeding, numerous fibrin patches and earlier onset of tumor formation with higher penetrance in animals with partial ablation of Dnmt3a in the hematopoietic compartment. Heightened colon pathology was reflected by modified murine endoscopic index of CAC severity (MEICS) (Fung and Putoczki 2018) (MEICS scores 7.5±2.0 in WT vs 10.0±3.2 in Dnmt3a +/-, p=0.05, n=14,13). We observed elevated incidence of tumors >2mm in diameter (40.2±22.2% in WT vs 74.2±15.7% in Dnmt3a +/-, p=0.03, in 5 independent trials) and a higher tumor burden in Dnmt3a +/- chimerae (7.4±7.8 mm 3 in WT vs 14.5±13.9 mm 3 in Dnmt3a +/-, p=0.0017, n=53,55) at end point. Histopathological analysis revealed marked immune infiltration, extensive ulceration and dysplasia of colonic epithelium, and more frequent adenocarcinoma formation with occasional submucosal invasion (histology score 20.2±4.4 in WT vs 24.6±6.9 in Dnmt3a +/-, p=0.03, n=10,9). To uncover signaling pathways deregulated by experimental Dnmt3a-driven CH, we surveyed the auto- and paracrine stimulatory loops by multiplex cytokine/chemokine assays and profiled transcriptomes of colon tumors by bulk RNA-seq. These studies identified gene signatures commonly associated with colon carcinogenesis including accentuated epithelial-to-mesenchymal transition (gene set enrichment analysis (GSEA) NES=2.42, padj=0.0096), Wnt/b-catenin (NES=2.07, padj=0.0096), and Vegf/angiogenesis (NES=1.83, padj=0.0096). Elevated proliferation (Ki67) and increased vascularization (CD31) were validated by immunohistochemistry. These aspects of tumor pathophysiology along with the composition and functional characteristics of the tumor-infiltrating leukocytes derived from animals with and without hematopoietic-specific Dnmt3a partial loss are being further investigated. Preliminary data showed bone marrow-derived macrophages (BMDMs) from Dnmt3a +/- chimera exhibit impaired ability to phagocytose syngeneic MC38 colon tumor cells, compared to WT. Our study provides new insights into the non-tumor-cell-autonomous mechanisms of carcinogenesis effected by alterations in the hematopoietic system and into the interplay between the immune system and the aggressive phenotype of colorectal cancer. Our findings may aid the development of novel therapeutic options to improve outcomes in solid tumor patients with CH. Figure 1 Figure 1. Disclosures Bowman: Mission Bio: Honoraria, Speakers Bureau. Levine: Ajax: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Zentalis: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Astellas: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Roche: Honoraria, Research Funding.

Tetraspanin CD82 Regulates Hematopoietic Stem and Progenitor Cell Quiescence and Regeneration

The significant cellular demand of the hematopoietic system is maintained by a rare pool of tissue-specific, hematopoietic stem and progenitor cells (HSPCs) that are primarily found in a quiescent state. Upon hemopoietic stresses, such as significant bleeding, overwhelming infection, and myelosuppressive therapy, HSPCs undergo rapid cell cycle activation, but ultimately must return to quiescence to prevent exhaustion of the hematopoietic system. Emerging evidence from our laboratory suggests that the tetraspanin CD82 plays a critical role in the regulation of HSPC quiescence and activation. Tetraspanins are membrane scaffold proteins with the ability to modulate signaling through the formation of tetraspanin-enriched microdomains, which organize membrane signaling receptors and intracellular signaling molecules critical for propagating downstream signaling. Previous data from our laboratory identified a role for CD82 in HSPC quiescence, where we find a reduction in long term-HSCs in global CD82KO mice, resulting from increased HSPC activation and a reduction of quiescent G 0 cells. In the present study, we test the hypothesis that CD82 expression promotes HSPC return to quiescence following hematopoietic stress, by regulating the activation of TGF-β signaling. To investigate the impact of CD82 expression on hematopoietic regeneration under stress, we treated WT and CD82KO mice with 2 doses of 200mg/kg of chemotherapy agent 5-FU and measured overall survival. Interestingly, we find that CD82KO mice have significantly longer overall survival compared to their WT counterparts. Moreover, we find an increase in peripheral blood HSPCs in CD82KO mice during the early recovery period from 5FU treatment suggestive of enhanced cell activation upon stress and effective regeneration post injury. Mechanistically, the multifunctional cytokine TGF-β plays an essential role in supporting HSPC quiescence and activation, as one of the most potent inhibitors of HSPC growth both in vitroand in vivo. To investigate whether CD82 plays a role in TGF-β signaling, we stimulated WT and CD82KO HSPCs with TGF-β and measured nuclear translocation of SMAD2/3, a downstream effector of TGF-β activation. Confocal imaging demonstrates that CD82KO HSPCs have decreased nuclear translocation of SMAD2/3 upon TGF-β activation, consistent with reduced TGF-β signaling. Similarly, primary human CD34 +HSPCs sorted for low and high CD82 expression also have decreased and increased SMAD2/3 nuclear translocation, respectively. Moreover, gene expression analysis of cell cycle regulators identified the reduced expression of p57, a downstream gene target of TGF-β signaling in HSPCs harvested from CD82KO mice. Currently, we are investigating the impact of the CD82 scaffold on TGF-β signaling of HSPCs localized within the context of the bone marrow niche, by analyzing bone marrow sections from mice injected with HSPC-like cells differentially expressing CD82. Together, these data suggest that CD82 regulates HSPC quiescence and activation through modulation of TGFβ signaling. Future studies will focus on investigating how CD82 modulates local TGFβ signaling within the niche. Disclosures No relevant conflicts of interest to declare.

Conditionally Pathogenic Genetic Variants of a Hematopoietic Disease-Suppressing Enhancer

Human genetic variants are classified based on potential pathogenicity to guide clinical decisions. However, mechanistic uncertainties often preclude definitive categorization. Germline coding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). The conserved murine enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy- and transplantation-induced hematopoietic regeneration in a cell-autonomous manner (Soukup et al., 2019). Since an epigenetically-silenced normal allele can exacerbate phenotypes of a pathogenic heterozygous variant, we engineered a bone marrow failure model harboring the Ets motif variant and a severe E-box:GATA enhancer mutation on the second allele (CH) that renders the enhancer chromatin inaccessible, to determine how perturbations of the hematopoietic system trigger pathogenesis. Given the propensity of GATA2 deficiency syndrome patients to infection, we asked if CH mice could mount an appropriate response to the bacterial cell wall component LPS. In WT, LPS acts on HSCs to induce inflammatory signaling and proliferation (Caiado et al., 2021). Twenty-four hours after LPS (0.5 mg/kg) administration, CH LSK levels were 3.1-fold lower than WT (p = 0.009). Immunophenotypic LT-HSCs (LSKCD150 +CD34 -) were 1.9-fold lower in treated CH vs. WT mice (p = 0.019). LPS reduced all LSK subpopulations: CD34 -CD150 -, CD34 +CD150 -, and CD34 +CD150 + were 3.4-, 5.7-, and 5.3-fold lower, respectively (p = 0.007, 0.029, and 0.006). Thus, the CH mutation attenuated acute inflammation-induced HSPC expansion. G-CSF is among the cytokines/chemokines induced by infection (Quinton et al., 2002). As G-CSF is used to mobilize bone marrow HSCs for collection prior to transplantation, we tested the following models: 1) +9.5 mutation abrogates HSPC expansion and/or mobilization in response to diverse stimuli; 2) +9.5 mutation permits expansion/mobilization, yet compromises function; 3) HSPC expansion, mobilization and function are +9.5 mutation-insensitive. After 8 doses of G-CSF, circulating neutrophil numbers increased 2.6-fold in WT (p = 0.003), while neutrophils did not increase in the CH mice. G-CSF treatment increased bone marrow HSCs 2.6-fold in WT mice, whereas the CH mutation abrogated this response (p < 0.0001). As a metric of mobilization, splenic HSCs were quantified. After G-CSF treatment, WT splenic HSCs expanded 11-fold (p < 0.0001), while CH HSCs were unchanged. To functionally analyze mobilized HSPCs, colony forming unit (CFU) assays were conducted with peripheral blood. CFU increased 11-fold in WT blood post-treatment (p < 0.0001), and CH abrogated the increase. To test whether G-CSF signaling is impacted by the CH mutation, we asked whether G-CSF treatment of bone marrow ex vivo induced STAT3 phosphorylation using a phospho-flow cytometry assay. While G-CSF induced pSTAT3 8.5-fold in WT Lin -Kit + cells (p < 0.0001), the induction was attenuated to 4.9-fold in CH (p = 0.003). Signaling in Gr1 + granulocytes was indistinguishable between genotypes (p = 0.999). Thus, the CH mutation attenuated G-CSF-induced signaling and HSPC mobilization, abrogated HSPC expansion, and compromised HSPC function. The Gata2 CH bone marrow failure model is characterized by relatively normal developmental hematopoiesis and adult steady-state hematopoiesis, yet the mice are vulnerable to stressors. As the CH mice were hypersensitive to myeloablation and acute inflammation and exhibited an attenuated response to a clinical HSC-mobilizing agent, GATA2-driven regenerative hematopoiesis mitigates damage resulting from chemotherapy and inflammation and promotes G-CSF-induced HSC mobilization. As the enhancer variant controlled HSC responsiveness to a therapeutically important HSC-mobilizing drug, these results link non-coding genome variation with mobilization efficacy. Based on the prominent GATA2 activities described herein, we are investigating how the full ensemble of macromolecular components constituting the GATA2 regulatory network generate a vital protective system conferring resilience to the hematopoietic system in the context of diverse stressors and whether genetic variants in genes encoding components of these pathways resemble or differ from those of GATA2 variants. Disclosures No relevant conflicts of interest to declare.

Evaluación del Método de Aprendizaje Basado en la Investigación (MABI) en el CICS UMA-IPN

El método basado en la investigación (MABI), emplea la Participación Referenciada y distintas técnicas de aprendizaje en el aula, apoyadas por las distintas herramientas didácticas, métodos educativos y los sistemas de información y comunicación, TIC´S. El objetivo de esta estrategia es mejorar y flexibilizar el Método de Aprendizaje Basado en la Investigación (MABI) a fin de que, no perdiendo su esencia, se adapte a las condiciones del docente y de los alumnos de los distintos currículos, a fin de elevar el nivel de aprendizaje de los alumnos. Este método se describe en la planeación didáctica del Programa académico: Médico Cirujano y Partero la Unidad de Aprendizaje: Ap. Cardiovascular y Sistema Hematopoyético, (Bioquímica) utilizado en la 41 Generación. Los resultados obtenidos fueron que el índice de reprobación de los alumnos disminuyo. Ya que aproximadamente el número de alumnos reprobados eran entre 10 o 12 alumnos por grupo en las generaciones pasadas en que solo se aplicaba un examen único y ahora después de aplicar este método el índice de reprobación es entre 2 y 4 alumnos reprobados por grupo de un total de 40 alumnos, obteniendo muy buenos resultados. Method Based Learning Research (MABI), Referenced method employs different techniques and learning in the classroom participation, supported by various teaching tools, educational methods and information and communication systems, TIC'S. The aim of this strategy is to improve and streamline Method Based Learning Research (MABI) so that, not losing its essence, it suits the conditions of teachers and students of different curricula, in order to raise the level of student learning. This method is described in educational planning the academic program: Surgeon and Obstetrician Learning Unit. Ap Cardiovascular and Hematopoietic System (Biochemistry) used in the 41 generation. The results were that the failure rate of students decreased. Since about the number of passing students were between 10 to 12 students per group in past generations that only a single exam and now after applying this method, the failure rate is between 2 and 4 students reproved by group is applied a total of 40 students, with very good results.

Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential

While many studies have described Drosophila embryonic and larval blood cells, the hematopoietic system of the imago remains poorly characterized and conflicting data have been published concerning adult hematopoiesis. Using a combination of blood cell markers, we show that the adult hematopoietic system is essentially composed of a few distinct mature blood cell types. In addition, our transcriptomics results indicate that adult and larval blood cells have both common and specific features and it appears that adult hemocytes reactivate many genes expressed in embryonic blood cells. Interestingly, we identify a small set of blood cells that does not express differentiation markers but rather maintains the expression of the progenitor marker domeMeso. Yet, we show that these cells are derived from the posterior signaling center, a specialized population of cells present in the larval lymph gland, rather than from larval blood cell progenitors, and that their maintenance depends on the EBF transcription factor Collier. Furthermore, while these cells are normally quiescent, we find that some of them can differentiate and proliferate in response to bacterial infection. In sum, our results indicate that adult flies harbor a small population of specialized cells with limited hematopoietic potential and further support the idea that no substantial hematopoiesis takes place during adulthood.