Unraveling T-cell Exhaustion: Genetic Screening Meets In Vitro Modeling.

T-cell exhaustion poses a significant barrier to the efficacy of immunotherapies. In the past decade, immune checkpoint blockade (ICB) has been the leading strategy to prevent or reverse T-cell exhaustion. Although ICB yields promising clinical outcomes in patients with cancer, its impact on T-cell reinvigoration is often short-lived. High-throughput genomic tools, including CRISPR screening along with single-cell RNA and chromatin accessibility sequencing may point toward new therapeutic avenues. However, their utility in identifying key mediators of T-cell exhaustion is constrained by the restricted scalability of well-validated in vivo exhaustion models, like chronic LCMV infection. In a recent article in Science Immunology, Wu and colleagues introduce an in vitro exhaustion model that involves repetitive stimulation of T-cell receptor-transgenic, LCMV-specific P14 CD8 T cells. This approach enables a direct comparison of exhausted T (Tex) cells generated both in vivo and in vitro using the same antigen, adeptly pinpointing exhaustion features that can be recapitulated in vitro. Leveraging this efficient and scalable model alongside CRISPR screening, the authors highlight the transcription factor BHLHE40 as a pivotal element in promoting Tex-cell transition from progenitor to intermediate Tex cells.

N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer.

A genome-wide PiggyBac transposon-mediated screen and a resistance screen in a PIK3CAH1047R-mutated murine tumor model reveal NF1 loss in mammary tumors resistant to the phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor alpelisib. Depletion of NF1 in PIK3CAH1047R breast cancer cell lines and a patient-derived organoid model shows that NF1 loss reduces sensitivity to PI3Kα inhibition and correlates with enhanced glycolysis and lower levels of reactive oxygen species (ROS). Unexpectedly, the antioxidant N-acetylcysteine (NAC) sensitizes NF1 knockout cells to PI3Kα inhibition and reverts their glycolytic phenotype. Global phospho-proteomics indicates that combination with NAC enhances the inhibitory effect of alpelisib on mTOR signaling. In public datasets of human breast cancer, we find that NF1 is frequently mutated and that such mutations are enriched in metastases, an indication for which use of PI3Kα inhibitors has been approved. Our results raise the attractive possibility of combining PI3Kα inhibition with NAC supplementation, especially in patients with drug-resistant metastases associated with NF1 loss.

Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy.

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.

Combination cancer immunotherapies: Emerging treatment strategies adapted to the tumor microenvironment.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment. However, resistance to ICB occurs frequently due to tumor-intrinsic alterations or extrinsic factors in the tumor microenvironment. This Viewpoint aims to give an update on recent developments in immunotherapy for solid tumors and highlights progress in translational research and clinical practice.

Protein Tyrosine Phosphatase SHP2 Controls Interleukin-8 Expression in Breast Cancer Cells.

Treatment of metastasis remains a clinical challenge and the majority of breast cancer-related deaths are the result of drug-resistant metastases. The protein tyrosine phosphatase SHP2 encoded by the proto-oncogene PTPN11 promotes breast cancer progression. Inhibition of SHP2 has been shown to decrease metastases formation in various breast cancer models, but specific downstream effectors of SHP2 remain poorly characterized. Certain cytokines in the metastatic cascade facilitate local invasion and promote metastatic colonization. In this study, we investigated cytokines affected by SHP2 that could be relevant for its pro-tumorigenic properties. We used a cytokine array to investigate differentially released cytokines in the supernatant of SHP2 inhibitor-treated breast cancer cells. Expression of CXCL8 transcripts and protein abundance were assessed in human breast cancer cell lines in which we blocked SHP2 using shRNA constructs or an allosteric inhibitor. The impact of SHP2 inhibition on the phospho-tyrosine-proteome and signaling was determined using mass spectrometry. From previously published RNAseq data (Aceto et al. in Nat. Med. 18:529-37, 2012), we computed transcription factor activities using an integrated system for motif activity response analysis (ISMARA) (Balwierz et al. in Genome Res. 24:869-84, 2014). Finally, using siRNA against ETS1, we investigated whether ETS1 directly influences CXCL8 expression levels. We found that IL-8 is one of the most downregulated cytokines in cell supernatants upon SHP2 blockade, with a twofold decrease in CXCL8 transcripts and a fourfold decrease in IL-8 protein. These effects were also observed in preclinical tumor models. Analysis of the phospho-tyrosine-proteome revealed that several effectors of the mitogen-activated protein kinase (MAPK) pathway are downregulated upon SHP2 inhibition in vitro. MEK1/2 inhibition consistently reduced IL-8 levels in breast cancer cell supernatants. Computational analysis of RNAseq data from SHP2-depleted tumors revealed reduced activity of the transcription factor ETS1, a direct target of ERK and a transcription factor reported to regulate IL-8 expression. Our work reveals that SHP2 mediates breast cancer progression by enhancing the production and secretion of the pro-metastatic cytokine IL-8. We also provide mechanistic insights into the effects of SHP2 inhibition and its downstream repercussions. Overall, these results support a rationale for targeting SHP2 in breast cancer.

Feed-forward loops between metastatic cancer cells and their microenvironment-the stage of escalation.

Breast cancer is the most frequent cancer among women, and metastases in distant organs are the leading cause of the cancer-related deaths. While survival of early-stage breast cancer patients has increased dramatically, the 5-year survival rate of metastatic patients has barely improved in the last 20 years. Metastases can arise up to decades after primary tumor resection, hinting at microenvironmental factors influencing the sudden outgrowth of disseminated tumor cells (DTCs). This review summarizes how the environment of the most common metastatic sites (lung, liver, bone, brain) is influenced by the primary tumor and by the varying dormancy of DTCs, with a special focus on how established metastases persist and grow in distant organs due to feed-forward loops (FFLs). We discuss in detail the importance of FFL of cancer cells with their microenvironment including the secretome, interaction with specialized tissue-specific cells, nutrients/metabolites, and that novel therapies should target not only the cancer cells but also the tumor microenvironment, which are thick as thieves.

Hepatic stellate cells suppress NK cell-sustained breast cancer dormancy.

The persistence of undetectable disseminated tumour cells (DTCs) after primary tumour resection poses a major challenge to effective cancer treatment1-3. These enduring dormant DTCs are seeds of future metastases, and the mechanisms that switch them from dormancy to outgrowth require definition. Because cancer dormancy provides a unique therapeutic window for preventing metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of reservoirs of dormant DTCs is imperative. Here we show that different tissue-specific microenvironments restrain or allow the progression of breast cancer in the liver-a frequent site of metastasis4 that is often associated with a poor prognosis5. Using mouse models, we show that there is a selective increase in natural killer (NK) cells in the dormant milieu. Adjuvant interleukin-15-based immunotherapy ensures an abundant pool of NK cells that sustains dormancy through interferon-γ signalling, thereby preventing hepatic metastases and prolonging survival. Exit from dormancy follows a marked contraction of the NK cell compartment and the concurrent accumulation of activated hepatic stellate cells (aHSCs). Our proteomics studies on liver co-cultures implicate the aHSC-secreted chemokine CXCL12 in the induction of NK cell quiescence through its cognate receptor CXCR4. CXCL12 expression and aHSC abundance are closely correlated in patients with liver metastases. Our data identify the interplay between NK cells and aHSCs as a master switch of cancer dormancy, and suggest that therapies aimed at normalizing the NK cell pool might succeed in preventing metastatic outgrowth.

The NFIB-ERO1A axis promotes breast cancer metastatic colonization of disseminated tumour cells.

Metastasis is the main cause of deaths related to solid cancers. Active transcriptional programmes are known to regulate the metastatic cascade but the molecular determinants of metastatic colonization remain elusive. Using an inducible piggyBac (PB) transposon mutagenesis screen, we have shown that overexpression of the transcription factor nuclear factor IB (NFIB) alone is sufficient to enhance primary mammary tumour growth and lung metastatic colonization. Mechanistically and functionally, NFIB directly increases expression of the oxidoreductase ERO1A, which enhances HIF1α-VEGFA-mediated angiogenesis and colonization, the last and fatal step of the metastatic cascade. NFIB is thus clinically relevant: it is preferentially expressed in the poor-prognostic group of basal-like breast cancers, and high expression of the NFIB/ERO1A/VEGFA pathway correlates with reduced breast cancer patient survival.