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Suppl. Fig. 1 confirms that ID8agg is more virulent versus parental ID8 in WT mice. Suppl. Fig. 2 provides relevant details for plasmids used to make recombinant cell lines, and gives Western blot validations for each PD-L1lo clone described. Suppl, Fig. 3 shows that αPD-L1 reduces tumor growth and metastatic spread of B16hi cells in NSG mice and shows color photomicrographs of lungs from treated mice. Suppl. Fig. 4 shows that αPD-L1 immunotherapy fails to treat ID8agg in wild-type mice. Suppl. Fig. 5 shows that tumor PD-L1 is necessary and sufficient for αPD-L1 treatment effects on B16 cells challenged into wild type mice. Suppl. Fig. 6 provides much additional Western blot data on mTORC1 and mTORC2 signals in B16 and ID8agg cells after rapamycin or chloroquine treatments. Suppl. Fig. 7 shows effects of tumor cell PD-L1 expression on B16 and ID8agg cell proliferation sensitivity to TNFα, cis-platinum and paclitaxel in vitro.

PD-L1 antibodies produce efficacious clinical responses in diverse human cancers, but the basis for their effects remains unclear, leaving a gap in the understanding of how to rationally leverage therapeutic activity. PD-L1 is widely expressed in tumor cells, but its contributions to tumor pathogenicity are incompletely understood. In this study, we evaluated the hypothesis that PD-L1 exerts tumor cell–intrinsic signals that are critical for pathogenesis. Using RNAi methodology, we attenuated PD-L1 in the murine ovarian cell line ID8agg and the melanoma cell line B16 (termed PD-L1lo cells), which express basal PD-L1. We observed that PD-L1lo cells proliferated more weakly than control cells in vitro. As expected, PD-L1lo cells formed tumors in immunocompetent mice relatively more slowly, but unexpectedly, they also formed tumors more slowly in immunodeficient NSG mice. RNA sequencing analysis identified a number of genes involved in autophagy and mTOR signaling that were affected by PD-L1 expression. In support of a functional role, PD-L1 attenuation augmented autophagy and blunted the ability of autophagy inhibitors to limit proliferation in vitro and in vivo in NSG mice. PD-L1 attenuation also reduced mTORC1 activity and augmented the antiproliferative effects of the mTORC1 inhibitor rapamycin. PD-L1lo cells were also relatively deficient in metastasis to the lung, and we found that anti-PD-L1 administration could block tumor cell growth and metastasis in NSG mice. This therapeutic effect was observed with B16 cells but not ID8agg cells, illustrating tumor- or compartmental-specific effects in the therapeutic setting. Overall, our findings extend understanding of PD-L1 functions, illustrate nonimmune effects of anti-PD-L1 immunotherapy, and suggest broader uses for PD-L1 as a biomarker for assessing cancer therapeutic responses. Cancer Res; 76(23); 6964–74. ©2016 AACR.

Although the APOBEC3 family of single-stranded DNA cytosine deaminases is well-known for its antiviral factors, these enzymes are rapidly gaining attention as prominent sources of mutation in cancer. APOBEC3′s signature single-base substitutions, C-to-T and C-to-G in TCA and TCT motifs, are evident in over 70% of human malignancies and dominate the mutational landscape of numerous individual tumors. Recent murine studies have established cause-and-effect relationships, with both human APOBEC3A and APOBEC3B proving capable of promoting tumor formation in vivo. Here, we investigate the molecular mechanism of APOBEC3A-driven tumor development using the murine Fah liver complementation and regeneration system. First, we show that APOBEC3A alone is capable of driving tumor development (without Tp53 knockdown as utilized in prior studies). Second, we show that the catalytic glutamic acid residue of APOBEC3A (E72) is required for tumor formation. Third, we show that an APOBEC3A separation-of-function mutant with compromised DNA deamination activity and wildtype RNA-editing activity is defective in promoting tumor formation. Collectively, these results demonstrate that APOBEC3A is a “master driver” that fuels tumor formation through a DNA deamination-dependent mechanism.

BackgroundTumor intracellular programmed cell death ligand-1 (PDL1) mediates pathologic signals that regulate clinical treatment responses distinctly from surface-expressed PDL1 targeted by αPDL1 immune checkpoint blockade antibodies.MethodsWe performed a drug screen for tumor cell PDL1 depleting drugs that identified Food and Drug Administration (FDA)-approved chlorambucil and also 9-[2-(phosphonomethoxy)ethyl] guanine. We used in vitro and in vivo assays to evaluate treatment and signaling effects of pharmacological tumor PDL1 depletion focused on chlorambucil as FDA approved, alone or plus αPDL1.ResultsPDL1-expressing mouse and human ovarian cancer lines and mouse melanoma were more sensitive to chlorambucil-mediated proliferation inhibition in vitro versus corresponding genetically PDL1-depleted lines. Orthotopic peritoneal PDL1-expressing ID8agg ovarian cancer and subcutaneous B16 melanoma tumors were more chlorambucil-sensitive in vivo versus corresponding genetically PDL1-depleted tumors. Chlorambucil enhanced αPDL1 efficacy in tumors otherwise αPDL1-refractory, and improved antitumor immunity and treatment efficacy in a natural killer cell-dependent manner alone and plus αPDL1. Chlorambucil-mediated PDL1 depletion was relatively tumor-cell selective in vivo, and treatment efficacy was preserved in PDL1KO hosts, demonstrating tumor PDL1-specific treatment effects. Chlorambucil induced PDL1-dependent immunogenic tumor cell death which could help explain immune contributions. Chlorambucil-mediated PDL1 reduction mechanisms were tumor cell-type-specific and involved transcriptional or post-translational mechanisms, including promoting PDL1 ubiquitination through the GSK3β/β-TRCP pathway. Chlorambucil-mediated tumor cell PDL1 depletion also phenocopied genetic PDL1 depletion in reducing tumor cell mTORC1 activation and tumor initiating cell content, and in augmenting autophagy, suggesting additional treatment potential.ConclusionsPharmacological tumor PDL1 depletion with chlorambucil targets tumor-intrinsic PDL1 signaling that mediates treatment resistance, especially in αPDL1-resistant tumors, generates PDL1-dependent tumor immunogenicity and inhibits tumor growth in immune-dependent and independent manners. It could improve treatment efficacy of selected agents in otherwise treatment-refractory, including αPDL1-refractory cancers, and is rapidly clinically translatable.

Regulatory T cells (Treg) engage IL-2 by high affinity CD25, and anti-tumor effector T cells (Teff) use intermediate affinity CD122. We studied IL-2 complexes (IL-2c) that selectively activate CD122+ Teff over CD25+ Tregs. We found IL-2c but not aPD-L1 potently inhibits ID8agg, an aggressive mouse ovarian cancer (OC) model. IL-2c decreased ascites Treg functional markers (CD25, GranzymeB) while upregulating them on Teffs. IL-2c inhibited Treg suppressive function in ascites but not TDLN. Ascites Tregs after IL-2c showed a fragile phenotype (increased PD-1, T-bet, and IFNg with maintained FoxP3) known to contribute to better immunotherapy response. These data suggest IL-2c inhibits Treg in the ascites through inducing fragile Tregs. Neither CD8+ T cells nor gd T cells is required for IL-2c induced Treg fragility. Studies testing other populations are ongoing. CD8+TCF-1+T cell stem cells (TCSC) improve immune checkpoint blockade in many cancer types. In ID8agg tumors, IL-2c induces CD8+TCF-1+ TCSC with tumor inhibitory capacity when transferred into Rag KO mice. tSNE analysis revealed IL-2c-induced TCSC are CXCR5+PD-1− express CCR2 CXCR3 and produce TNFa, while aPD-L1 induced TCSC are CXCR5+ and PD-1+, consistent with previous reports. These data suggest IL-2c induces beneficial CD8+TCSC distinct from aPD-L1. Although aPD-L1 is not efficacious alone in ID8agg, it significantly promoted IL-2c efficacy. Mice treated with IL-2c+αPD-L1 are resistant to tumor re-challenge even 6 weeks after final dose, suggesting durable immune memory. In sum, IL-2c is a novel OC immunotherapy that targets distinct immune pathways simultaneously, including inhibiting Tregs, boosting TCSCs, and inducing immune memory when combined with aPD-L1.

Tumor PD-L1 mediates non-canonical, intracellular signals including the DNA damage response (DDR), of unclear significance and mechanisms. We show that genetic PD-L1 depletion (PD-L1KO) destabilized the Chk2 protein, a DNA damage response (DDR) factor, resulting in ATM/Chk2 pathway defects but not ATR/Chk1 in melanoma, bladder, breast, and ovarian cancer. Consistent with ATM/Chk2 defects, PD-L1 KO led to DNA damage (γH2AX) and impaired homologous recombination (HR) (p-RPA32, BRCA1, Rad51 foci). Thus, PD-L1 KO vs control cells were significantly more sensitive to DDR inhibitors (DDRi) against ATR, Chk1, and PARP in vitro and in vivo in NSG mice. Chk2 regulation by PD-L1 was independent of PD-L1 cytoplasmic tail yet required intracellular (vs surface) PD-L1 suggesting physical PD-L1/Chk2 interaction supported by IP and imaging. PD-L1 stabilized Chk2 protein by preventing its lysosomal degradation without altering Chek2 mRNA. αPD-L1 is thought to work by protecting PD-L1 induced anti-tumor T cell suppression via PD-1, but PD-L1 DDR effects were PD-1-independent. Intracellular PD-L1 suppressed DDRi induced cGAS/STING activation by immunoblots and qRT-PCR of type 1 IFN genes. In vivo in WT mice, genetic PD-L1 depletion but not αPD-L1, sensitized highly immunotherapy resistant 4T1 breast cancer to PARPi. Strikingly, PARPi had reduced effect on PD-L1KO tumors in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi efficacy. Our work implicates a novel role of intracellular PD-L1 in DDR and tumor immunogenicity and identifies related therapeutic vulnerabilities exposed by intracellular PD-L1 targeting. Surface vs intracellular PD-L1, and specific DDR signals could be treatment response biomarkers.

PD-L1 has become a major target in anti-cancer immunotherapy, however the overall response rate still remains relatively low among most types of cancers, notably breast cancer. There is an unmet need to boost efficacy of immune checkpoint blockade therapies. While both tumor and host immune cell-derived PD-L1 are implicated in immune suppressive functions of PD-L1, the exact immunosuppressive contribution of PD-L1 from different host tissues is little studied. Here we show that PD-L1 expression is significantly higher in human breast adipose tissue versus stromal vascular fractions. In vitro adipogenesis of mouse pre-adipocytes significantly up-regulates PD-L1 versus pre-adipocytes. In vitro co-culture shows that adipocyte PD-L1 suppresses T cell activation and response to anti-PD-L1. In an adipocyte-specific knockout (KO) mouse model, we show that syngeneic mammary tumors grow slower in KO than wildtype hosts. Immunophenotyping shows that tumors grown in KO mice have higher CD8+and CD4+ T cell infiltration as well as more CD8+ Prf+ cytotoxic T cells. Tumor tissue RNA-seq analysis reveals that genetic ablation of adipocyte PD-L1 confers a distinct transcriptomic signature of T cell activation and tumor killing. Our current findings uncover a previously unappreciated source of immune suppressive PD-L1 in the breast cancer microenvironment and could inform novel therapeutic strategies through targeting tumor-surrounding adipose tissue for treating breast cancer.

Tumor PD-L1 mediates diverse cell-intrinsic signals that increase cancer pathogenesis including the DNA damage response (DDR). Although immune consequences of tumor-extrinsic PD-L1 blockade are known, novel therapeutic vulnerabilities from inhibiting tumor-intrinsic PD-L1 signals are little studied. To assess tumor-intrinsic PD-L1 DDR control, we depleted PD-L1 by CRISPR/Cas9 or shRNA (PD-L1KO) in mouse B16 (melanoma), 4T1 (breast), ID8agg (ovarian), and human bladder RT4 cancer lines. We identified specific DDR signaling defects in PD-L1KO versus control (con) cells after exposure to distinct DNA damaging agents. 24h gemcitabine (or 2 Gy X-ray) in vitro in PD-L1KO vs. con RT4 cells elevated γH2AX (DNA damage marker) and reduced Chk2 DDR gene expression but not ATM, ATR, Chk1, or BRCA1 (immunoblots), and decreased BRCA1/p-RPA32 nuclear foci formation (confocal) altogether suggesting tumor-intrinsic PD-L1 promotes specific DDR/DNA repair pathways. PD-L1 loss could thus predict synthetic lethality to DDR inhibitors (DDRi) Chk1i or PARPi. PD-L1KORT4, ID8, and 4T1 cells were more sensitive to small molecule Chk1i in vitro (>15 fold vs. con, MTT) and in vivo in NSG mice. Chk1i and PARPi led to γH2AX increase in PD-L1KO B16, RT4, and 4T1 vs. sham treated tumor cells as predicted by DDR defects. Genetic cellular PD-L1 depletion, but not anti-PD-L1 (extrinsic), strongly sensitized B16 and 4T1 tumors to PARPi in vivo. Strikingly, PARPi had no effect on PD-L1KO B16 in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi treatment efficacy. Thus, DDRi plus inhibiting tumor intrinsic PD-L1 signals could improve immunotherapy in immunotherapy-sensitive or resistant tumors.

αPD-L1 bladder cancer (BC) immunotherapy is effective in

Aging is the biggest risk factor for cancer, yet little is known about how it affects cancer immunotherapy. Melanoma response to αPD-1/αPD-L1 correlates with CD8+ TCF-1+ T cell stem cell (TCSC) generation. We tested αPD-1 (100 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18–24 months) and young (3–8 months) mice challenged orthotopically with B16 melanoma (SQ) or ID8agg ovarian cancer (IP). Tumors and draining lymph nodes (TDLN) were analyzed by flow. We previously reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young. αPD-L2 treated B16 in aged but not young, the first single agent cancer immunotherapy exhibiting this property. Preliminary data indicate survival benefit of αPD-L2 against ID8agg in aged but not young mice. Aged mice tolerated all treatments comparable to young. B16 efficacy in young (αPD-1, αPD-L1) and aged (αPD-1, αPD-L2) correlated with increased TCSC and total TIL, but TCSC differed by age and treatment. PD-1 interacts with PD-L1 and PD-L2 while PD-L1 interacts with PD-1 and CD80. Aged mice expressed significantly more T-cell PD-1 and PD-L2 and up to 40-fold more PD-L2, PD-L1 and CD80 vs. young on myeloid cells and NK cells. We generated bone marrow-derived DC with GM-CSF. Aged DC expressed much high PD-L2 versus young, suggesting a cell-intrinsic PD-L2 age bias. Treatment differences in aged versus young could depend on these immune checkpoint or TCSC differences. We are now identifying mechanisms for increased PD-L2 and contributions to αPD-L2 efficacy in aged, and testing TCSC effects on treatments. Our work can improve cancer immunotherapy in aged hosts and provide models for treatment resistance in young hosts.

We have shown that cancer cell-intrinsic PD-L1 signals increase mTORC1 and unbundle actin. To test if tumor cell PD-L1 affects the DNA damage response (DDR) we made PD-L1KO mouse ID8agg (ovarian) and B16 (melanoma), and human bladder RT4 cancer lines. The DNA damaging agent gemcitabine (gem) reduced PD-L1KO ID8agg and RT4 viability in vitro vs. control (con) (ID8agg: 29X vs. 1.3X; RT4: 2X vs 1.1X). 4h gem increased phospho (p)-γH2AX, a marker of DNA double strand breaks: 2X in PD-L1KO vs. con RT4. P-γH2AX stayed abnormally high after gem removal, further showing defective DDR in PD-L1KO. 24h gem also induced more p-γH2AX in PD-L1KO vs. con ID8agg, and 48h gem induced more apoptosis in PD-L1KO vs. con RT4. Cisplatin, a DNA damaging agent distinct from gem, reduced PD-L1KO ID8agg viability 9X vs. 2X in con. No viability difference was seen in cisplatin treated PD-L1KO vs. con RT4. Thus, PD-L1 chemo resistance effects are cell type and/or agent specific. To define DDR mechanism(s), we found that 24h gem decreased p-Chk2 (ATM DDR pathway), due to reduced total Chk2, but not p-Chk1 or total Chk1 (ATR DDR pathway) in PD-L1KO vs. con RT4. 20 Gy X-rays (DNA damage distinct vs. gem), reduced p-Chk1 in PD-L1KO but not con B16 and induced less p-RPA32 in PD-L1KO vs. con B16, suggesting defects specifically in homologous recombination mediated DNA repair. Thus, cell-intrinsic PD-L1 DDR control is insult and/or cell type specific. RT4 DDR effects were independent of mTOR or actin. DDR affects immunotherapy so PD-L1 cell-intrinsic DDR control could be exploited in treatment or response biomarkers. DDR inhibitors, chemo or radiation combos could improve anti-PD-L1 efficacy in distinct cancers, subjects of ongoing work, as is testing tumor cell intrinsic PD-1 effects.

Tumor-intrinsic PD-L1 signals contribute to tumor virulence and pathology, but mechanisms are poorly understood. We previously showed that tumor-intrinsic PD-L1 increases mTORC1 and tumor initiating cell (TIC) generation and function in ovarian cancer and melanoma. We used CRISPR/Cas9 (KO) and shRNA (lo) to reduce PD-L1 in mouse melanoma (B16) and ovarian cancer (ID8agg), and human ovarian cancer (ES2). PD-L1lo reduced mTORC1 and TIC as expected, but unexpectedly increased actin polymerization and filopodia, and altered cell morphology. The actin depolymerizing agents Latrunculin-A or Cytochalasin-D reversed actin polymerization in PD-L1lo ID8agg cells and increased mTORC1 (p-rpS6) without affecting mTORC2 (pAktS473). Latrunculin-A and Cytochalasin-D increased TIC numbers and stemness gene expression (Oct4) in PD-L1lo ID8agg cells with no effect on control ID8agg cells. Similar actin effects were seen in PD-L1KO B16 and PD-L1KO ES2. To confirm TIC function, Latrunculin-A increased tumorospheres (TIC self-renewal function) in PD-L1lo, but not control ID8agg cells. The mTORC1 inhibitor rapamycin reduced TIC numbers and functions with no effect on actin polymerization, suggesting mTORC1 is downstream of actin polymerization. Rptorlo ID8agg cells (with low mTORC1 signaling) did not exhibit the PD-L1lo actin polymerization, nor did Latrunculin-A increase Rptorlo ID8agg TIC numbers, tumorospheres, or stemness genes, confirming the flow of signals is PD-L1 to actin to mTORC1 to TIC (stemness). Our data define a highly novel tumor virulence control mechanism of cell-intrinsic PD-L1 through inhibiting actin polymerization suggesting new drug discovery targets. Preliminary data show some similar effects of tumor PD-1.

αPD-L1 is an FDA-approved bladder cancer (BC) immunotherapy but is effective in ≤30% of cases. We tested conjugates of αIL-2 antibody + IL-2 (IL-2c) that block IL-2 from binding high-affinity IL-2Rα (CD25) for preferential IL-2Rβ(CD122) binding. CD25 and CD122 are preferred for IL-2 capture by regulatory T cells and anti-tumor effector T cells (Teff), respectively, allowing IL-2c to target Teff preferentially. Orthotopic, intravesical (in bladder) MB49 BC produces PD-L1+BC tumors in syngeneic BL6 mice. αPD-L1 or IL-2c treated intravesical, but not lung metastatic MB49 produced by intravenous injection. Still, in metastatic BC, αPD-L1 + IL-2c (combo) reduced lung metastases and extended survival. Preliminary data found combo treatment efficacy was better in lung versus bladder. In subcutaneous (SQ) B16 melanoma, combo was better than single agents, and increased CD8+CXCR5+TCF-1+Tim-3−PD-1+T stem cells (CXCR5+SC, see X. Zhang poster) vs. single agents. Neither αPD-L1 nor combo increased CXCR5+SC in MB49 in bladder or lung metastases, suggesting a novel treatment mechanism. To test the impact of tumor PD-L1 on treatment efficacy, we made PD-L1KOMB49, but it did not grow in bladder or SQ in wild type mice, but grew similar to control MB49 in immunodeficient NSG mice SQ, suggesting tumor PD-L1 microenvironment-specific effects. PD-L1KOB16 grew well SQ in wild type mice, excluding a PD-L1-specific defect. Thus, tumor PD-L1 differs in immune evasion in a tumor-dependent manner. Selective IL-2 targeting to CD122 improves αPD-L1 treatment of metastatic BC. Mechanisms differ from melanoma, which could be due to tumor, PD-L1, or microenvironment effects. We are assessing mechanisms and αPD-1 treatment effects (also FDA-approved for BC).

Tumors express intracellular PD-L1 that we showed increases ovarian cancer (OC) and melanoma virulence by increasing mTORC1 and tumor stem cells (TSC). We made PD-L1KO(CRISPR) in ES2 human OC and B16 melanoma cells and re-expressed PD-L1 only on cell surface (PD-L1surf) or in cytoplasm (PD-L1cyto) with specific expression vectors. Subcellular PD-L1 location in all clones was validated by immunoblots, confocal microscopy, and co-IP/mass spec. We selected clones with PD-L1 surf or cyto expression similar to parental cells. PD-L1KO reduced cell diameter 30% which was restored partially in PD-L1surf and fully in PD-L1cyto in ES2 and B16. PD-L1KO significantly decreased viability in all cell lines tested which was fully rescued in PD-L1surf and PD-L1cyto. PD-L1surf ES2 and B16 grew slower in vitro and in vivo than PD-L1cyto and only PD-L1cyto rescued F-actin polymerization phenotype seen in PD-L1KO ES2 and B16. RNA-seq showed that surf and cyto PD-L1 differentially altered canonical signaling pathways, notably mTOR, stemness and gene regulation. PD-L1cyto grew much slower than PD-L1surf B16 in NSG mice but generated more TSC in vitro, and PD-L1surf but not PD-L1cyto B16 responded to αPD-L1 in WT. PD-L1cyto but not PD-L1surf induced mTORC1 activation (p-rpS6, p-P70S6K) through mTORC1 lysosomal docking through LAMTOR1/2 seen by confocal and confirmed by co-IP in ES2 and B16. Imaging CyTOF of human melanomas showed PD-L1cyto association with activated mTOR. Thus, surf versus cyto PD-L1 differentially affects tumor biology, signaling, pathology, TIL and treatment response in OC and melanoma. We define a mechanism for PD-L1-driven mTORC1 activation. Surface vs. cytoplasmic PD-L1 and/or mTORC1 could be response biomarkers for αPD-L1 or αPD-1.

We reported that tumor intrinsic PD-L1 promotes tumor initiating cell (TIC) virulence and mTORC1. Others have focused on PI3K/AKT/mTOR in promoting TIC, but specific downstream mTOR regulators of tumor stemness are unknown. We knocked down Raptor (mTORC1 component, Rptorlo) in ID8agg cells (murine ovarian cancer). Rptorlo cells had reduced mTORC1 with no change in PD-L1 in total cells or CD44+CD24+ TIC. %TIC was reduced in Rptorlo cell cultures, and Rptorlo TIC were functionally defective for self-renewal by tumorosphere formation. Rapamycin (mTORC1 inhibitor) reduced ID8agg Rptor, tumorospheres, and the stemness and virulence genes Oct4 and Nanog. Oct4 knockin to PD-L1lo ID8agg restored TIC numbers and spheres. Together, data confirm PD-L1 drives stemness through Oct4 by increasing mTORC1. Rptorlo TIC formed significantly smaller tumors vs. control TIC in immunodeficient NSG mice, confirming TIC intrinsic Rptor drives immune independent virulence. In wild type mice, Rptorlo vs. control TIC produced less ascites and smaller tumors with lower %TIC in vivo (but similar PD-L1), significantly more recently-activated CD44hiCD62LloCD8+ cells in ascites and draining lymph nodes (DLN). CCR2+CD8+ T cells and CXCR5+TCF1+PD-1+Tim3− stem-like T cells were also increased in Rptorlo DLN. Thus, Rptorlo TIC influence systemic and tumor-associated immunity. In high grade serous ovarian tumors (The Cancer Genome Atlas) high Rptor expression predicted a significantly higher cancer stem cell signature, and lower median survival in late stage patients. Our work establishes tumor intrinsic Rptor driven by PD-L1 plays a key role in regulating TIC and stem like T cells and can be a prognostic marker for late stage ovarian cancer patients.

The antiretroviral therapy (ART) significantly increased the number of aging HIV patients and the non-AIDS defining cancers (NADC) have become the most frequent co-morbidity and causes of death among HIV patients. The role of HIV infection in cancer initiation and progression is unclear. Tumor initiating cells (TIC) are responsible for chemo-resistance, metastasis and mortality. Our previous work demonstrated that tumor-intrinsic PD-L1 correlate with proportion (%) of TIC and virulence. Supernatants from HIV infected peripheral blood lymphocyte (PBMC) cultures (HIV-sup) significantly increased % TIC (CD44+PD-L1+) in two human tumor cell lines-SCC4 (squamous carcinoma) and HeLa (cervical cancer). HIV single stranded RNA (ssRNA) binds TLR7/8, and leads to immune activation and inflammation. The Cancer Genome Atlas database analyses showed that squamous cancer and cervical adenocarcinoma patients with TLR7/8 amplification had significantly poor overall survival. TLR7 agonist R848 led to significant increase in % TIC, whereas treatment of the cancer cells with HIV-sup in presence of TLR7/8 inhibitor ODN2088 reversed this effect. Similarly, pharmacological inhibition (by ACHP) of TLR7/8 mediated NFkB signaling pathway decreased TIC % two-fold, confirming HIV mediated TLR7/8 signaling has pro-TIC effects. High levels of inflammatory cytokine IFNα correlates with HIV load and persistent infection. Treatment with IFNα led to a significant increase in TIC whereas blocking IFNα-induced JAK signaling by ruxolitinib, in presence of HIV-sup reduced TIC significantly. Thus, our novel data gives insight into HIV induced host-pathogen interactions that lead to TIC prevalence.

Anti-tumor T cells (Teffs) engage IL2 largely through CD122, vs. CD25 for regulatory T cells (Tregs). In ID8agg ovarian cancer (OC), CD122-selective IL2 complexes (IL2c, made from CD122-blocking ab+IL-2), increased Teff numbers and functions as expected but unexpectedly reduced tumor Treg function, with near-complete tumor rejection in OC. IL2c reduced lineage specific molecules (e.g., Tbet) and cytokines (e.g., IFNg) in tumor Tregs, suggesting IL2c improves Treg differentiation, but not explaining IL2c-driven Treg defects. In vitro, IL-2 and IL-2c produced similar Treg numbers and Foxp3 from naïve CD4+T cells, with functional data pending. αPD-L1 improved IL2c efficacy for full tumor rejection in OC and B16 melanoma. Increased CD8+CXCR5+PD1+Tim3−TCF-1+stem-like T cells (CXCR5+) are associated with favorable αPD-1 or αPD-L1 outcome in mice and humans. IL2c + αPD-L1 markedly increased CXCR5+in B16 melanoma and to a lesser extent in OC. IL2c + αPD-L1 increased tumor CXCR5+perforin expression in B16 vs. either single agent. Injecting tumor-infiltrating CXCR5+into RagKO mice produced CXCR5+Tim3− and CXCR5−Tim3+cells confirming tumor CXCR5+stem cell properties. These data demonstrate several novel IL2c mechanisms to exploit in distinct cancers and treatment combinations. Ongoing work tests IL2c, αPD-L1 and combo treatment effects on CXCR5+ stemness and mechanisms for CXCR5+ induction, and mechanisms for IL2c-driven Treg dysfunction.

PD-L1, expressed on many tumors, inhibits anti-tumor T cells by programmed death (PD)-1 engagement. Tumor PD-L1 predicts anti-PD-L1 treatment outcomes through incompletely understood mechanisms. We used naturally PD-L1+ murine B16 melanoma (ctrl) and made PD-L1KO B16 by CRISPR/Cas9. PD-L1KO B16 grew faster when ctrl B16 was on the opposite flank (trans). By contrast, ctrl B16 grew slower when trans to PD-L1KO B16. Anti-PD-L1 slowed ctrl but not PD-L1KO B16 tumors as expected. However, when PD-L1KO was trans to ctrl αPD-L1 now also slowed PD-L1KO growth. Anti-PD-L1 increased CD3+ T cell infiltration into ctrl and PD-L1KO tumors similarly in vivo, but increased natural killer (NK) cell numbers and functions (e.g., CD107a, IFN-g) in PD-L1KO tumors > ctrl. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly > ctrl, but NK cells migrated 2-fold more to PD-L1KO vs ctrl. PD-L1KO tumors produced more CCL2 than ctrl in vitro and in vivo, and this CCL2 induced CCR2+ NK chemotaxis in vitro. Both NK cells and T cells were required for optimal trans responses to anti-PD-L1 treatment in vivo. In wild type mice, anti-CCL2 reduced growth of trans PD-L1KO tumors > ctrl, and reduced anti-PD-L1 efficacy against PD-L1KO> ctrl tumors. In CCR2KO mice, anti-PD-L1 efficacy was retained against trans ctrl B16, but abolished against PD-L1KO trans tumors. PD-L1 also regulated other chemokines recruiting receptor+ cells, with similar effects seen in MB49 bladder cancer and 4T1 breast cancer cells. Thus, tumor PD-L1 alters immune infiltrates and anti-PD-L1 efficacy through novel chemokine effects. Our models help understand anti-PD-L1 (and likely anti-PD-1) responses based on tumor PD-L1 expression and can define ways to improve immunotherapy for PD-L1 null tumors.

The programmed death protein 1 (PD-1) and ligand (PD-L1) axis inhibits T cell activation and reduces anti-tumor immunity. Most studies of the PD-1/PD-L1 axis test extrinsic, immune-dependent effects. However, tumor cell-intrinsic PD-L1 regulates immune-independent tumor mTOR signals, autophagy, and tumor initiating cell generation. Melanoma cell-intrinsic PD-1 regulates melanoma growth and mTOR. We tested PD-1 expression and induction in multiple human tumor cell lines. We detected PD-1 in human cell lines in vitro from ES2 ovarian cancer, VMM122 melanoma, and RT4 bladder cancer by flow cytometry, Western blot and RT-PCR. All lines were negative for the other PD-L1 receptor, CD80, by flow cytometry. Incubating lines with IFNα or IFNγ for 48 hours further augmented PD-1 expression as detected by flow, Western blot and RT-PCR. αPD-1 antibody (50 μg/ml) reduced proliferation of RT4 bladder cancer cells in vitro, demonstrating a direct PD-1 signal effect in these cells, and αPD-L1 similarly reduced in vitro proliferation. Interestingly, anti-PD-1 effects on reducing proliferation were similar: 41.5% reduction; p

PD-1/PD-L1 provides a mechanism of immune escape, the blockage of which has reinvigorated interest in the treatment of urothelial cancer. We recently reported on cell-intrinsic PD-L1 effects in melanoma and ovarian cancer, and considered that effects in bladder cancer cells could differ based on distinct mutational landscapes. Using CRISPER/Cas9 methodology, we knockout PD-L1 in the murine bladder cancer cell line MB49 and the human bladder caner cell line RT. We show here that cell-intrinsic MB49 and RT4 cells express PD-L1 and PD-1 that each mediate cell-intrinsic signals. PD-L1 knockout has little effect on cells proliferation in vitro, but significant difference could be found in tumor size when we used MB49 Ctrl or MB49 PD-L1KO cells to challenge C57B16 mice. αPD-L1 and αPD-1 antibodies reduced bladder cancer cell proliferation in vitro demonstrating direct signaling effects. Bladder cancer cell-intrinsic PD-L1 promoted mTORC1 and tumor initiating cell generation similar to melanoma and ovarian cancer cells. By contrast to melanoma and ovarian cancer cells, bladder cancer cell PD-L1 promoted autophagy and had little effect on in vivo immune-independent growth. Base one the functional role, PD-L1 knockout increased proliferation inhibition induced by Rapamycin in both mouse and human bladder cancer cells. Furthermore, we found intrinsic PD-L1 suppress cytokine production, including CXCL10. Corresponding with increased CXCL10 secretion, more CXCR3+ NK cells could be attracted by PD-L1KO cancer cells. Overall, our findings further illustrated the intrinsic role of PD-L1 in bladder cancer development and found PD-1 expression on human bladder cancer cells for the first time.

We reported that PD-L1 regulates tumor cell-intrinsic mTOR, autophagy and tumor initiating cell (TIC) generation. We found significant cytoplasmic PD-L1 in various mouse and human tumor cell lines by Western, flow cytometry and confocal. To assess subcellular localized PD-L1 effects, we CRISPR knocked out PD-L1 (KO) from mouse B16 melanoma cells and re-expressed PD-L1 only on surface (surfKI; Display vector) or cytoplasm (cytoKI; PcDNA vector). KO cells proliferated slowly vs ctrl in vitro, partially restored by surfKI and cytoKI. KO suppressed mTORC1 (p70S6K), which was reversed by cytoKI but not surfKI. Rapamycin (mTORC1 inhibitor) reduced surfKI and KO proliferation equally, but both > cytoKI (57% vs 88%, p

TIC mediate tumor formation, drug resistance and relapse. We reported that PD-L1 promotes tumor mTORC1, and TIC number and functions. Knocking down PD-L1 reduced expression of stemness genes Oct4 and Nanog and the mTORC1 signaling component Raptor in B16 melanoma and ID8agg ovarian cancer (OC) cells. To understand how PD-L1 controls stemness, we treated control and PD-L1 KO (KO) B16 cells with rapamycin (mTORC1 inhibitor), PI3K inhibitor wortmannin, Notch inhibitor Compound E, and c-Myc inhibitor JQ1. Only rapamycin affected control and KO CD133+CD44+CD24+ TIC differentially (50% reduced TIC in control versus 0% in KO, p=0.0075). We over-expressed Oct4 in B16 but failed to get clones, so over-expressed it in ID8agg PD-L1lo cells (KI) and found that TICs (CD44+CD24+) had improved proliferation and 2-fold more TIC than PD-L1lo cells (p=0.015, p=0.018). We sorted ID8agg, PD-L1lo and KI TIC and cultured them in stem cell medium. KI TICs formed four-fold bigger spheres than PD-L1lo TIC (p

Tumor B7-H1 (PD-L1) promotes tumor immune evasion by interaction with T cell PD-1. B16 melanoma and ID8agg ovarian cancer express B7-H1. We genetically made B7-H1lo or B7-H1hi lines to study additional B7-H1 effects. Control B16 and ID8agg grew more rapidly in vitro versus B7-H1lo cells and B7-H1hi B16 cells grew faster than control. In vivo, B16 and ID8agg grew faster than B7-H1lo lines in immunodeficient NSG mice. B16 challenge in NSG mice with B7-H1hi cells produced more lung metastases versus parental, which produced more metastases than B7-H1lo. αB7-H1 significantly reduced tumor growth and metastases. These data support an immune-independent role for tumor B7-H1 in tumor growth and metastatic spread. Control B16 and ID8agg cells had elevated TORC1 and reduced TORC2 versus B7-H1lo cells. The mTOR inhibitor rapamycin reduced cell growth greater in parental versus B7-H1lo cells in vitro. Also in line with reduced TORC1, B7-H1lo cells had increased autophagy (LC3-II/actin ratio and autophagosome formation) compared to parental cells. Notably, autophagy inhibitors chloroquine and 3-methyladenine had a greater inhibitory effect on in vitro proliferation of control B16 versus B7-H1lo cells. Inhibition of autophagy in vivo with either agent significantly reduced tumor growth of B16 but not B7-H1lo cells, suggesting that B7-H1 replete tumors are more dependent on the stress-modulating function of autophagy versus B7-H1lo tumors. Our studies show novel tumor-intrinsic B7-H1 signal effects that regulate mTOR and autophagy signals directly associated with immune-independent tumor growth, supporting a paradigm shifting concept that tumor B7-H1 augments stress response and metabolism through cell-intrinsic mechanisms.

Cancer stem cells (CSC) resist chemotherapy and radiation, leading to treatment failures and reduced patient survival. Thus, CSC are clinically relevant therapy targets. B7-H1 is an immune checkpoint molecule and a target for αB7-H1 immunotherapy. We generated control (native B7-H1+) and B7-H1lo B16 melanoma lines by shRNA. B16*ctrl cultures were 4.2% CSCs (CD44+CD133+CD24+) vs. 0.19% in B16*B7-H1lo. CSC from B16*B7-H1lo cultures formed fewer and smaller (