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Introduction Mounting evidence suggest that macrophages play crucial roles in disease and tissue regeneration. However, despite much efforts during the past decade, our knowledge about the extent of macrophages' contribution to adult pancreatic regeneration after injury or during pancreatic disease progression is still limited. Nevertheless, it is generally accepted that some macrophage features that normally would contribute to healing and regeneration may be detrimental in pancreatic cancer. Altogether, the current literature contains conflicting reports on whether macrophages act as friends or foe in these conditions. Methods and Results In this review, we briefly review the origins of tissue resident and infiltrating macrophages and the importance of cellular crosstalking between macrophages and other resident cells in tissue regeneration. The primary objective of this review is to summarize our knowledge of the distinct roles of tissue resident and infiltrating macrophages, the impact of M1 and M2 macrophage phenotypes, and emerging evidence on macrophage crosstalking in pancreatic injury, regeneration, and disease. Conclusion Macrophages are involved with various stages of pancreatic cancer development, pancreatitis, and diabetes. Elucidating their role in these conditions will aid the development of targeted therapeutic treatments.

Diffuse midline glioma is an aggressive brain tumor with a median age at diagnosis of 6.3 years and 5-year survival rate of 2.2%.The defining histone mutation H3K27M precludes docking of a protein responsible for epigenetic modification resulting in erroneously accessible chromatin and subsequent transcription of oncogenic pathways, including the RAS-MERK5-ERK5 signaling cascade. To determine which oncogenic processes are regulated by extracellular signal-regulated kinase 5 (ERK5), gene-set enrichment analysis of patient-derived datasets demonstrated gene networks involved in glycolysis to be enriched with ERK5 expression. In confirmation, loss of ERK5 via shRNA interference reduced cell proliferation and glycolysis in DIPG IV and SF8628 cells. Reintroduction of ERK5 wildtype (WT) into ERK5 knockdown lines rescued cell proliferation and glycolysis, while the addition of ERK5 kinase dead domain (KDD) only partially rescued these survival and metabolic defects. Targeted evaluation of glycolysis enzyme expression via qRT-PCR revealed a direct relationship between ERK5 and proglycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Mechanistically, ERK5 activation of PFKFB3 was mediated by activation of transcriptional factor myocyte enhancer factor 2A (MEF2A) as demonstrated by coimmunoprecipitation and luciferase promoter assays. Expression of PFKFB3 was elevated at both the mRNA and protein level in DMG patient-derived samples. Genetic knockdown of PFKFB3 via shRNA interference resulted in reduced proliferation and glycolysis in DIPG IV and SF8628 cells. Similarly, pharmacologic inhibition of PFKFB3 with small molecule inhibitor PFK-158 capitulated these results. This inhibitor demonstrated blood brain barrier penetrance in silico and extended survival of in vivo mouse models. Multitargeted drug therapy against both ERK5 and PFKFB3 produced a synergetic in vitro response with increased sensitivity of these cells to apoptosis compared to single treatment alone. In conclusion, these results support ERK5 regulation of glycolysis through the critical metabolic effector PFKFB3. Multitargeted drug therapy against this axis represents a therapeutic vulnerability in pediatric diffuse midline glioma.

In the version of this article initially published, the Acknowledgements statement should have included the following: "S.C.M. is

Diffuse Midline gliomas (DMGs) are grade IV tumors by the World Health Organization. They are inoperable and resistant to chemo/radiotherapies resulting in a median survival of 8-11 months and a 5-year survival of <2%. DMG is an epigenetic disease characterized by mutations on histone H3.3 K27M resulting in global transcriptional reprogramming. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model using clinically relevant co-alterations in Olig2+ neural progenitor cells (NPCs). Using an unbiased systems biology approach, we identified a reliance of H3K27M but not isogenic controls to the amino acid methionine, and the enzymes methionine adenosyltransferase 2A (MAT2A), and adenosylmethionine decarboxylase 1 (AMD1). MAT2A is a master regulator of methionine metabolism that converts methionine into the universal methyl donor S-adenosylmethionine (SAM) which is later converted into decarboxylated SAM (dcSAM) by AMD1 for polyamine metabolism. We postulated that targeting methionine regulator MAT2A through genetic/pharmacological abrogation would selectively alter DMG viability by disrupting the methylome. We discovered a novel mechanism demonstrating H3K27M cells are sensitive to MAT2A loss independent of methylthioadenosine phosphorylase (MTAP) deletions but rather through AMD1 overexpression. The current paradigm shows that MAT2A protein expression is inversely correlated with cellular SAM concentrations as sensed by splicing complex and m6A reader methyltransferase-like protein 16 (METTL16). To investigate the molecular mechanism by which H3K27M represses MAT2A, we postulated that dcSAM, the resultant metabolite of AMD1, promote(s) high turnover of METTL16–MAT2A transcript interactions like SAM, thereby diminishing MAT2A transcript and protein expression. We found that exogenous dcSAM promoted MAT2A intron retention and lower mature transcript levels. Our findings demonstrate that H3K27M leads to increased AMD1 protein expression resulting in diminished MAT2A expression. Combinatorial treatments inhibiting MAT2A and AMD1 may presents exploitable therapeutic vulnerabilities in these gliomas.

BACKGROUND High-grade gliomas (HGGs) are the most common fatal intrinsic brain tumors in pediatric patients. H3K27-altered diffuse midline gliomas (H3K27-DMGs), a subgroup of HGGs defined by a histone 3 position 27 alteration, are especially aggressive and result in the poorest patient outcomes. Despite in-depth genomic characterization, the 5-year survival rate has yet to improve beyond 2% following diagnosis. A common feature of H3K27-DMGs is infiltration of microglia, macrophages, other myeloid cells, collectively referred to as GAMs, and a small population of T-cells. The contribution of non-tumor cells in the tumor microenvironment (TME) can both promote and or inhibit tumor growth, thus representing an opportunity in the pursuit of novel therapeutics. Using bioinformatic analysis on a human H3K37-DMG single cell-RNA sequencing dataset, we reveal several cell-to-cell communication signaling networks, mediated by ligand and receptor pairs, between GAMs and tumor cells, respectively. HYPOTHESIS Microglial-derived growth factors activate oncogenic signaling pathways via paracrine signaling axes, thus promoting H3K27-DMG tumor cell proliferation and growth. METHODS I will validate these findings and test their therapeutic potential using co-culture studies, CRISPR and shRNA gene silencing, and phospho-proteomics technology. RELEVANCE This research provides further insights on the contribution of non-tumor cells in the TME towards H3K27-DMG cell proliferation and growth and could potentially inform future therapy paradigms.

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H3K27-mutant diffuse midline gliomas (DMGs) are defined as grade IV tumors by the World Health Organization. DMGs are inoperable and resistant to chemo/radio therapies. Median survival ranges from 8-11 months, with 2% of patients surviving beyond 5 years. H3K27M mutations lead to global epigenetic and transcriptional reprogramming driven by global loss of negative transcriptional regulator H3K27 trimethylation (H3K27me3). Loss of H3K27me3 is an initiating event in gliomagenesis. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model. An unbiased integrated systems biology approach identified that H3K27M but not isogenic controls relied on the amino acid methionine and the enzyme Methionine Adenosyltransferase 2A (MAT2A). MAT2A is a central regulator of one-carbon metabolism by converting methionine to S-adenosylmethionine (SAM), the universal methyl-donor for protein and nucleotide methylation reactions. In complementary genetic approaches, we applied these findings to patient-derived cell lines with the H3K27M mutation. We hypothesize that MAT2A abrogation, genetic/pharmacological, would alter DMG viability by disrupting the methylome. The current MAT2A sensitivity paradigm is based on Methylthioadenosine Phosphorylase (MTAP) deletion through a synthetic lethal mechanism. We provide a novel mechanism whereby H3K27M cells are sensitive to MAT2A loss, independent of MTAP and through Adenosylmethionine Decarboxylase 1 (AMD1) overexpression disrupting MAT2A regulation. This results in H3K27M cells having lower MAT2A protein levels, conferring a sensitivity by inhibiting residual MAT2A. Genetic/pharmacological aberrations to MAT2A resulted in reduced proliferation. Parallel H3K36me3 ChIP and RNA-sequencing identified loss of oncogenic and developmental transcriptional programs associated with MAT2A loss. In vivo syngeneic and patient-derived xenograft models with both inducible MAT2A knockdown or methionine restricted diets showed extended survival. These results suggest novel interactions between methionine metabolism and the epigenome of H3K27M gliomas and provide evidence that MAT2A, presents exploitable therapeutic vulnerabilities in histone mutant gliomas.