Targeting branched N-glycans and fucosylation sensitizes ovarian tumors to immune checkpoint blockade.

Aberrant glycosylation is a crucial strategy employed by cancer cells to evade cellular immunity. However, it's unclear whether homologous recombination (HR) status-dependent glycosylation can be therapeutically explored. Here, we show that the inhibition of branched N-glycans sensitizes HR-proficient, but not HR-deficient, epithelial ovarian cancers (EOCs) to immune checkpoint blockade (ICB). In contrast to fucosylation whose inhibition sensitizes EOCs to anti-PD-L1 immunotherapy regardless of HR-status, we observe an enrichment of branched N-glycans on HR-proficient compared to HR-deficient EOCs. Mechanistically, BRCA1/2 transcriptionally promotes the expression of MGAT5, the enzyme responsible for catalyzing branched N-glycans. The branched N-glycans on HR-proficient tumors augment their resistance to anti-PD-L1 by enhancing its binding with PD-1 on CD8+ T cells. In orthotopic, syngeneic EOC models in female mice, inhibiting branched N-glycans using 2-Deoxy-D-glucose sensitizes HR-proficient, but not HR-deficient EOCs, to anti-PD-L1. These findings indicate branched N-glycans as promising therapeutic targets whose inhibition sensitizes HR-proficient EOCs to ICB by overcoming immune evasion.

Acetate acts as a metabolic immunomodulator by bolstering T-cell effector function and potentiating antitumor immunity in breast cancer.

Acetate metabolism is an important metabolic pathway in many cancers and is controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer is well described, the consequences of blocking tumor acetate metabolism on the tumor microenvironment and antitumor immunity are unknown. We demonstrate that blocking ACSS2, switches cancer cells from acetate consumers to producers of acetate thereby freeing acetate for tumor-infiltrating lymphocytes to use as a fuel source. We show that acetate supplementation metabolically bolsters T-cell effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or a small-molecule inhibitor promotes an antitumor immune response and enhances the efficacy of chemotherapy in preclinical breast cancer models. We propose a paradigm for targeting acetate metabolism in cancer in which inhibition of ACSS2 dually acts to impair tumor cell metabolism and potentiate antitumor immunity.

The African-centric P47S Variant of TP53 Confers Immune Dysregulation and Impaired Response to Immune Checkpoint Inhibition.

The tumor suppressor TP53 is the most frequently mutated gene in cancer and is mutationally inactivated in 50% of sporadic tumors. Inactivating mutations in TP53 also occur in Li Fraumeni syndrome (LFS). In addition to germline mutations in TP53 in LFS that completely inactivate this protein, there are many more germline mutant forms of TP53 in human populations that partially inactivate this protein: we call these partially inactivating mutations "hypomorphs." One of these hypomorphs is a SNP that exists in 6%-10% of Africans and 1%-2% of African Americans, which changes proline at amino acid 47 to serine (Pro47Ser; P47S). We previously showed that the P47S variant of p53 is intrinsically impaired for tumor suppressor function, and that this SNP is associated with increased cancer risk in mice and humans. Here we show that this SNP also influences the tumor microenvironment, and the immune microenvironment profile in P47S mice is more protumorigenic. At basal levels, P47S mice show impaired memory T-cell formation and function, along with increased anti-inflammatory (so-called "M2") macrophages. We show that in tumor-bearing P47S mice, there is an increase in immunosuppressive myeloid-derived suppressor cells and decreased numbers of activated dendritic cells, macrophages, and B cells, along with evidence for increased T-cell exhaustion in the tumor microenvironment. Finally, we show that P47S mice demonstrate an incomplete response to anti-PD-L1 therapy. Our combined data suggest that the African-centric P47S variant leads to both intrinsic and extrinsic defects in tumor suppression. Significance: Findings presented here show that the P47S variant of TP53 influences the immune microenvironment, and the immune response to cancer. This is the first time that a naturally occurring genetic variant of TP53 has been shown to negatively impact the immune microenvironment and the response to immunotherapy.

An African-Specific Variant of TP53 Reveals PADI4 as a Regulator of p53-Mediated Tumor Suppression.

TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified. Here, we characterize a rare, African-specific germline variant of TP53 in the DNA-binding domain Tyr107His (Y107H). Nuclear magnetic resonance and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this, we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the nonnatural amino acid citrulline. Surprisingly, we show that Y107H mice develop spontaneous cancers and metastases and that Y107H shows impaired tumor suppression in two other models. We show that PADI4 is itself tumor suppressive and that it requires an intact immune system for tumor suppression. We identify a p53-PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. SIGNIFICANCE: We analyze the African-centric Y107H hypomorphic variant and show that it confers increased cancer risk; we use Y107H in order to identify PADI4 as a key tumor-suppressive p53 target gene that contributes to an immune modulation signature and that is predictive of cancer survival and the success of immunotherapy. See related commentary by Bhatta and Cooks, p. 1518. This article is highlighted in the In This Issue feature, p. 1501.

Targeting the mevalonate pathway suppresses ARID1A-inactivated cancers by promoting pyroptosis.

ARID1A, encoding a subunit of the SWI/SNF complex, is mutated in ∼50% of clear cell ovarian carcinoma (OCCC) cases. Here we show that inhibition of the mevalonate pathway synergizes with immune checkpoint blockade (ICB) by driving inflammasome-regulated immunomodulating pyroptosis in ARID1A-inactivated OCCCs. SWI/SNF inactivation downregulates the rate-limiting enzymes in the mevalonate pathway such as HMGCR and HMGCS1, which creates a dependence on the residual activity of the pathway in ARID1A-inactivated cells. Inhibitors of the mevalonate pathway such as simvastatin suppresses the growth of ARID1A mutant, but not wild-type, OCCCs. In addition, simvastatin synergizes with anti-PD-L1 antibody in a genetic OCCC mouse model driven by conditional Arid1a inactivation and in a humanized immunocompetent ARID1A mutant patient-derived OCCC mouse model. Our data indicate that inhibition of the mevalonate pathway simultaneously suppresses tumor cell growth and boosts antitumor immunity by promoting pyroptosis, which synergizes with ICB in suppressing ARID1A-mutated cancers.

Dual CD4-based CAR T cells with distinct costimulatory domains mitigate HIV pathogenesis in vivo.

An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4+ T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4+ T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.

Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity.

BACKGROUND: The gp41 component of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) contains a long cytoplasmic domain (CD) with multiple highly conserved tyrosine (Y) and dileucine (LL) motifs. Studies suggest that the motifs distal to major endocytosis motif (Y712HRL), located at residues 712-715 of Env, may contribute to Env functionality in the viral life cycle. In order to examine the biological contribution of these motifs in the biosynthesis, transport, and function of Env, we constructed two panels of mutants in which the conserved Y- and LL-motifs were sequentially substituted by alternative residues, either in the presence or absence of Y712. Additional mutants targeting individual motifs were then constructed. RESULTS: All mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C) contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells. CONCLUSIONS: From the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.