Pharmacological tumor PDL1 depletion with chlorambucil treats ovarian cancer and melanoma: improves antitumor immunity and renders anti-PDL1-resistant tumors anti-PDL1-sensitive through NK cell effects.

BACKGROUND: Tumor 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. METHODS: We 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. RESULTS: PDL1-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 GSK3ß/ß-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. CONCLUSIONS: Pharmacological 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.

Tumor Intrinsic PD-L1 Promotes DNA Repair in Distinct Cancers and Suppresses PARP Inhibitor-Induced Synthetic Lethality.

BRCA1-mediated homologous recombination is an important DNA repair mechanism that is the target of FDA-approved PARP inhibitors, yet details of BRCA1-mediated functions remain to be fully elucidated. Similarly, immune checkpoint molecules are targets of FDA-approved cancer immunotherapies, but the biological and mechanistic consequences of their application are incompletely understood. We show here that the immune checkpoint molecule PD-L1 regulates homologous recombination in cancer cells by promoting BRCA1 nuclear foci formation and DNA end resection. Genetic depletion of tumor PD-L1 reduced homologous recombination, increased nonhomologous end joining, and elicited synthetic lethality to PARP inhibitors olaparib and talazoparib in vitro in some, but not all, BRCA1 wild-type tumor cells. In vivo, genetic depletion of tumor PD-L1 rendered olaparib-resistant tumors sensitive to olaparib. In contrast, anti-PD-L1 immune checkpoint blockade neither enhanced olaparib synthetic lethality nor improved its efficacy in vitro or in wild-type mice. Tumor PD-L1 did not alter expression of BRCA1 or its cofactor BARD1 but instead coimmunoprecipitated with BARD1 and increased BRCA1 nuclear accumulation. Tumor PD-L1 depletion enhanced tumor CCL5 expression and TANK-binding kinase 1 activation in vitro, similar to known immune-potentiating effects of PARP inhibitors. Collectively, these data define immune-dependent and immune-independent effects of PARP inhibitor treatment and genetic tumor PD-L1 depletion. Moreover, they implicate a tumor cell-intrinsic, immune checkpoint-independent function of PD-L1 in cancer cell BRCA1-mediated DNA damage repair with translational potential, including as a treatment response biomarker. SIGNIFICANCE: PD-L1 upregulates BRCA1-mediated homologous recombination, and PD-L1-deficient tumors exhibit BRCAness by manifesting synthetic lethality in response to PARP inhibitors, revealing an exploitable therapeutic vulnerability and a candidate treatment response biomarker. See related commentary by Hanks, p. 2069.

CD122-targeted interleukin-2 and αPD-L1 treat bladder cancer and melanoma via distinct mechanisms, including CD122-driven natural killer cell maturation.

Bladder cancer (BC) and melanoma are amenable to immune checkpoint blockade (ICB) therapy, yet most patients with advanced/metastatic disease do not respond. CD122-targeted interleukin (IL)-2 can improve ICB efficacy, but mechanisms are unclear. We tested αPD-L1 and CD122-directed immunotherapy with IL-2/αIL-2 complexes (IL-2c) in primary and metastatic bladder and melanoma tumors. IL-2c treatment of orthotopic MB49 and MBT-2 BC generated NK cell antitumor immunity through enhanced activation, reduced exhaustion, and promotion of a mature, effector NK cell phenotype. By comparison, subcutaneous B16-F10 melanoma, which is IL-2c sensitive, requires CD8+ T and not NK cells, yet we found αPD-L1 efficacy requires both CD8+ T and NK cells. We then explored αPD-L1 and IL-2c mechanisms at distinct metastatic sites and found intraperitoneal B16-F10 metastases were sensitive to αPD-L1 and IL-2c, with IL-2c but not αPD-L1, increasing CD122+ mature NK cell function, confirming conserved IL-2c effects in distinct cancer types and anatomic compartments. αPD-L1 failed to control tumor growth and prolong survival in B16-F10 lung metastases, yet IL-2c treated B16-F10 lung metastases effectively even in T cell and adaptive immunity deficient mice, which was abrogated by NK cell depletion in wild-type mice. Flow cytometric analyses of NK cells in B16-F10 lung metastases suggest that IL-2c directly boosts NK cell activation and effector function. Thus, αPD-L1 and IL-2c mediate nonredundant, immune microenvironment-specific treatment mechanisms involving CD8+ T and NK cells in primary and metastatic BC and melanoma. Mechanistic differences suggest effective treatment combinations including in other tumors or sites, warranting further studies.

CD122-directed interleukin-2 treatment mechanisms in bladder cancer differ from αPD-L1 and include tissue-selective γδ T cell activation.

BACKGROUND: Anti-programmed death-ligand 1 (αPD-L1) immunotherapy is approved to treat bladder cancer (BC) but is effective in <30% of patients. Interleukin (IL)-2/αIL-2 complexes (IL-2c) that preferentially target IL-2 receptor ß (CD122) augment CD8+ antitumor T cells known to improve αPD-L1 efficacy. We hypothesized that the tumor microenvironment, including local immune cells in primary versus metastatic BC, differentially affects immunotherapy responses and that IL-2c effects could differ from, and thus complement αPD-L1. METHODS: We studied mechanisms of IL-2c and αPD-L1 efficacy using PD-L1+ mouse BC cell lines MB49 and MBT-2 in orthotopic (bladder) and metastatic (lung) sites. RESULTS: IL-2c reduced orthotopic tumor burden and extended survival in MB49 and MBT-2 BC models, similar to αPD-L1. Using antibody-mediated cell depletions and genetically T cell-deficient mice, we unexpectedly found that CD8+ T cells were not necessary for IL-2c efficacy against tumors in bladder, whereas γδ T cells, not reported to contribute to αPD-L1 efficacy, were indispensable for IL-2c efficacy there. αPD-L1 responsiveness in bladder required conventional T cells as expected, but not γδ T cells, altogether defining distinct mechanisms for IL-2c and αPD-L1 efficacy. γδ T cells did not improve IL-2c treatment of subcutaneously challenged BC or orthotopic (peritoneal) ovarian cancer, consistent with tissue-specific and/or tumor-specific γδ T cell contributions to IL-2c efficacy. IL-2c significantly altered bladder intratumoral γδ T cell content, activation status, and specific γδ T cell subsets with antitumor or protumor effector functions. Neither IL-2c nor αPD-L1 alone treated lung metastatic MB49 or MBT-2 BC, but their combination improved survival in both models. Combination treatment efficacy in lungs required CD8+ T cells but not γδ T cells. CONCLUSIONS: Mechanistic insights into differential IL-2c and αPD-L1 treatment and tissue-dependent effects could help develop rational combination treatment strategies to improve treatment efficacy in distinct cancers. These studies also provide insights into γδ T cell contributions to immunotherapy in bladder and engagement of adaptive immunity by IL-2c plus αPD-L1 to treat refractory lung metastases.

Bladder cancer cell-intrinsic PD-L1 signals promote mTOR and autophagy activation that can be inhibited to improve cytotoxic chemotherapy.

Tumor cell-intrinsic programmed death-ligand 1 (PD-L1) signals mediate immunopathologic effects in breast, colon, and ovarian cancers and in melanomas, but bladder cancer (BC) effects are unreported. We show here that BC cell-intrinsic PD-L1 signals in mouse MB49 and human RT4, UM-UC3, and UM-UC-14 BC cells regulate important pathologic pathways and processes, including effects not reported in other cancers. α-PD-L1 antibodies reduced BC cell proliferation in vitro, demonstrating direct signaling effects. BC cell-intrinsic PD-L1 promoted mammalian target of rapamycin complex 1 (mTORC1) signals in vitro and augmented in vivo immune-independent cell growth and metastatic cancer spread, similar to effects we reported in melanoma and ovarian cancer. BC cell-intrinsic PD-L1 signals also promoted basal and stress-induced autophagy, whereas these signals inhibited autophagy in melanoma and ovarian cancer cells. BC cell-intrinsic PD-L1 also mediated chemotherapy resistance to the commonly used BC chemotherapy agents cis-platinum and gemcitabine and to the mTORC1 inhibitor, rapamycin. Thus, BC cell-intrinsic PD-L1 signals regulate important virulence and treatment resistance pathways that suggest novel, actionable treatment targets meriting additional studies. As a proof-of-concept, we showed that the autophagy inhibitor chloroquine improved cis-platinum treatment efficacy in vivo, with greater efficacy in PD-L1 null versus PD-L1-replete BC.

CD122-Selective IL2 Complexes Reduce Immunosuppression, Promote Treg Fragility, and Sensitize Tumor Response to PD-L1 Blockade.

The IL2 receptor (IL2R) is an attractive cancer immunotherapy target that controls immunosuppressive T regulatory cells (Treg) and antitumor T cells. Here we used IL2Rß-selective IL2/anti-IL2 complexes (IL2c) to stimulate effector T cells preferentially in the orthotopic mouse ID8agg ovarian cancer model. Despite strong tumor rejection, IL2c unexpectedly lowered the tumor microenvironmental CD8+/Treg ratio. IL2c reduced tumor microenvironmental Treg suppression and induced a fragile Treg phenotype, helping explain improved efficacy despite numerically increased Tregs without affecting Treg in draining lymph nodes. IL2c also reduced Treg-mediated, high-affinity IL2R signaling needed for optimal Treg functions, a likely mechanism for reduced Treg suppression. Effector T-cell IL2R signaling was simultaneously improved, suggesting that IL2c inhibits Treg functions without hindering effector T cells, a limitation of most Treg depletion agents. Anti-PD-L1 antibody did not treat ID8agg, but adding IL2c generated complete tumor regressions and protective immune memory not achieved by either monotherapy. Similar anti-PD-L1 augmentation of IL2c and degradation of Treg functions were seen in subcutaneous B16 melanoma. Thus, IL2c is a multifunctional immunotherapy agent that stimulates immunity, reduces immunosuppression in a site-specific manner, and combines with other immunotherapies to treat distinct tumors in distinct anatomic compartments. SIGNIFICANCE: These findings present CD122-targeted IL2 complexes as an advancement in cancer immunotherapy, as they reduce Treg immunosuppression, improve anticancer immunity, and boost PD-L1 immune checkpoint blockade efficacy in distinct tumors and anatomic locations.

Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer.

As tumor PD-L1 provides signals to anti-tumor PD-1+ T cells that blunt their functions, αPD-1 and αPD-L1 antibodies have been developed as anti-cancer immunotherapies based on interrupting this signaling axis. However, tumor cell-intrinsic PD-L1 signals also regulate immune-independent tumor cell proliferation and mTOR signals, among other important effects. Tumor initiating cells (TIC) generate carcinomas, resist treatments and promote relapse. We show here that in murine B16 melanoma and ID8agg ovarian carcinoma cells, TIC express more PD-L1 versus non-TIC. Silencing PD-L1 in B16 and ID8agg cells by shRNA ("PD-L1lo") reduced TIC numbers, the canonical TIC genes nanog and pou5f1 (oct4), and functions as assessed by tumorosphere development, immune-dependent and immune-independent tumorigenesis, and serial transplantability in vivo. Strikingly, tumor PD-L1 sensitized TIC to interferon-γ and rapamycin in vitro. Cell-intrinsic PD-L1 similarly drove functional TIC generation, canonical TIC gene expression, and sensitivity to interferon-γ and rapamycin in human ES2 ovarian cancer cells. Thus, tumor-intrinsic PD-L1 signals promote TIC generation and virulence, possibly by promoting canonical TIC gene expression, suggesting that PD-L1 has novel signaling effects on cancer pathogenesis and treatment responses.

Immunotherapy for ovarian cancer.

OPINION STATEMENT: All work referenced herein relates to treatment of epithelial ovarian carcinomas, as their treatment differs from ovarian germ cell cancers and other rare ovarian cancers, the treatments of which are addressed elsewhere. Fallopian tube cancers and primary peritoneal adenocarcinomatosis are also generally treated as epithelial ovarian cancers. The standard of care initial treatment of advanced stage epithelial ovarian cancer is optimal debulking surgery as feasible plus chemotherapy with a platinum plus a taxane agent. If this front-line approach fails, as it too often the case, several FDA-approved agents are available for salvage therapy. However, because no second-line therapy for advanced-stage epithelial ovarian cancer is typically curative, we prefer referral to clinical trials as logistically feasible, even if it means referring patients outside our system. Immune therapy has a sound theoretical basis for treating carcinomas generally, and for treating ovarian cancer in particular. Advances in understanding the immunopathogenic basis of ovarian cancer, and the immunopathologic basis for prior failures of immunotherapy for it and other carcinomas promises to afford novel treatment approaches with potential for significant efficacy, and reduced toxicities compared with cytotoxic agents. Thus, referral to early phase immunotherapy trials for ovarian cancer patients that fail conventional treatment merits consideration.

Effect of serum estradiol and leptin levels on thyroid function, food intake and body weight gain in female Wistar rats.

We evaluated the interplay among estrogen, leptin and thyroid function in the regulation of body mass in female rats. Adult female rats were divided into four groups: control (C, sham-operated), ovariectomized (OVX), ovariectomized treated with estradiol benzoate (Eb) 0.7 or 14microg/100gbw per day, during 21 days. OVX led to an increase in body mass, food intake and food efficiency (change in body mass as function of the amount of food ingested) which were normalized by the lower Eb dose, and decreased significantly when the higher dose was given. Serum leptin levels were increased more than two-fold in all ovariectomized groups. Serum T4 levels of the Eb treated OVX were significantly lower than in the controls. Serum T3 and TSH were unaffected by OVX or by Eb treatment. Uterine type 2 iodothyronine deiodinase (D2) activity changed in parallel with serum estradiol: decreased after OVX, returned to control levels after the lower E2 treatment, and increased significantly after the high Eb dosage. The hypothalamic D2 activity was reduced around 30% in all castrated groups, treated or not with estrogen, whereas in the brown adipose tissue the enzyme was not changed. Interestingly, although estrogen-treated OVX rats had lower body weight, serum leptin was high, suggesting that estrogen increases leptin secretion. Our results show that estradiol is necessary for the hypothalamic action of leptin, since the increase in leptin levels observed in all ovariectomized rats was associated with a decrease in food intake and food efficiency only in the rats treated with estrogen.