SUMMARY Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

Summary Most patients with advanced triple-negative breast cancer (TNBC) develop drug resistance. MYC and MCL1 are frequently co-amplified in drug-resistant TNBC after neoadjuvant chemotherapy. Herein, we demonstrate that MYC and MCL1 cooperate in the maintenance of chemotherapy-resistant cancer stem cells (CSCs) in TNBC. MYC and MCL1 increased mitochondrial oxidative phosphorylation (mtOXPHOS) and the generation of reactive oxygen species (ROS), processes involved in maintenance of CSCs. A mutant of MCL1 that cannot localize in mitochondria reduced mtOXPHOS, ROS levels and drug-resistant CSCs without affecting the anti-apoptotic function of MCL1. Increased levels of ROS, a by-product of activated mtOXPHOS, led to the accumulation of HIF-1α. Pharmacological inhibition of HIF-1α attenuated CSC enrichment and tumor initiation in vivo. These data suggest that 1) MYC and MCL1 confer resistance to chemotherapy by expanding CSCs via mtOXPHOS; and 2) targeting mitochondrial respiration and HIF-1α may reverse chemotherapy resistance in TNBC.

provided expertise to develop 18 F nutrient uptake assays. F.X. and M.N.T injected and handled mice for 18 F nutrient uptake assays, and performed and provided expertise for PET imaging and autoradiography. T.H. and W.D.M. performed and provided expertise for intrarenal Renca experiments. R.W.J. and V.T.M generated and provided expertise for PyMT GEMM tumors. R.E.B and C.S.W. generated and provided expertise for AOM/DSS CRC tumors. B.I.R. R.T.O. and M.H.W. generated the pTZeo-EL-thy1.1 transposon construct and engineered MC38 cells using this transposon system. B.I.R, M.Z.M, and A.S. performed in vivo 2NBDG studies. J.E.B. provided expertise in characterizing TAM. A.R.P provided expertise in flow sorting for mRNA transcript analysis. B.I.R. and M.Z.M performed extracellular flux and mRNA transcript experiments. F.M.M. and E.F.M performed and provided expertise in cell staining for light microscopy. E.F.M performed light microscopy and pathologic examination of MC38 tumors. A.A (VU) conducted transcriptomic analysis. B.I.R and M.Z.M. analyzed all data generated in this study. J.C.R. and W.K.R. obtained funding for this study.Data Availability Statement (DAS) All data will be made available upon reasonable request to JCR/WKR. Tumor mRNA transcript data that support the findings of this study have been deposited in Gene Expression Omnibus (GEO) under accession GSE165223. These data are also found in Supplementary Information Table 4. Code Availability Statement (CAS)The code used to support tumor mRNA transcript analysis has been previously published (see methods references) and will be made available upon request to JCR/WKR.

Renal cell carcinoma (RCC) comprises a diverse group of malignancies arising from the nephron. The most prevalent type, clear cell renal cell carcinoma (ccRCC), is characterized by genetic mutations in factors governing the hypoxia signaling pathway, resulting in metabolic dysregulation, heightened angiogenesis, intratumoral heterogeneity, and deleterious tumor microenvironmental (TME) crosstalk. Identification of specific genetic variances has led to therapeutic innovation and improved survival for patients with ccRCC. Current barriers to effective long-term therapeutic success highlight the need for continued drug development using improved modeling systems. ccRCC preclinical models can be grouped into three broad categories: cell line, mouse, and 3D models. Yet, the breadth of important unanswered questions in ccRCC research far exceeds the accessibility of model systems capable of carrying them out. Accordingly, we review the strengths, weaknesses, and therapeutic implications of each model system that are relied upon today.

Summary Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.

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Purpose: Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of ex vivo-engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor.Experimental Design: The role of PIM kinases in T cells was studied either by genetic ablation (PIM1 À/À PIM2 À/À PIM3 À/À ) or its pharmacologic inhibition (pan-PIM kinase inhibitor, PimKi). Murine melanoma B16 was established subcutane-ously and treated by transferring tumor epitope gp100-reactive T cells along with treatment regimen that involved inhibiting PIM kinases, anti-PD1 or both.Results: With inhibition of PIM kinases, T cells had significant reduction in their uptake of glucose, and upregulated expression of memory-associated genes that inversely correlate with glycolysis. In addition, the expression of CD38, which negatively regulates the metabolic fitness of the T cells, was also reduced in PimKi-treated cells. Importantly, the efficacy of antitumor T-cell therapy was markedly improved by inhibiting PIM kinases in tumor-bearing mice receiving ACT, and further enhanced by adding anti-PD1 antibody to this combination.Conclusions: This study highlights the potential therapeutic significance of combinatorial strategies where ACT and inhibition of signaling kinase with checkpoint blockade could improve tumor control.

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