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Summary: Much work has been done to reduce cancer immunosuppression through inhibiting soluble proteins, surface molecules, and suppressive cells. This article shows an important role for the lipid lysophosphatidic acid, whose suppression shows promise as a novel cancer immunotherapeutic, demonstrated in ovarian cancer. See related article by Chae et al., 1904 (5).

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

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies. Significance: This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy. See related commentary by Osorio and Zamarin, p. 766

Despite repeated associations between T cell infiltration and outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that the hallmarks of tumor recognition in ovarian cancer-infiltrating T cells are primarily restricted to tissue-resident memory (TRM) cells. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3(+) CD8(+)CD103(+)CD69(+) TRM cells and immunohistochemistry of 122 high-grade serous ovarian cancers shows that only progenitor (TCF1(low)) tissue-resident T cells (TRMstem cells), but not recirculating TCF1(+) T cells, predict ovarian cancer outcome. TRMstem cells arise from transitional recirculating T cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes that eventually become exhausted. Therefore, ovarian cancer is indeed an immunogenic disease, but that depends on similar to 13% of CD8(+) tumor-infiltrating T cells (similar to 3% of CD8(+) clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM-like cells. Our results define the signature of relevant tumor-reactive T cells in human ovarian cancer, which could be applicable to other tumors with unideal mutational burden.

Purpose: Immunotherapy treats some cancers, but not ovarian cancer. Regulatory T cells (Tregs) impede anti-ovarian cancer immunity but effective human Treg-directed treatments are lacking. We tested Treg depletion with denileukin diftitox (DD) ± IFNα as ovarian cancer immunotherapy. Patients and Methods: Mice with syngeneic ID8 ovarian cancer challenge were treated with DD, IFNα, or both. The phase 0/I trial tested one dose-escalated DD infusion for functional Treg reduction, safety, and tolerability. The phase II trial added IFNα2a to DD if DD alone failed clinically. Results: DD depleted Tregs, and improved antitumor immunity and survival in mice. IFNα significantly improved antitumor immunity and survival with DD. IFNα did not alter Treg numbers or function but boosted tumor-specific immunity and reduced tumor Treg function with DD by inducing dendritic cell IL6. DD alone was well tolerated, depleted functional blood Tregs and improved immunity in patients with various malignancies in phase 0/I. A patient with ovarian cancer in phase 0/I experienced partial clinical response prompting a phase II ovarian cancer trial, but DD alone failed phase II. Another phase II trial added pegylated IFNα2a to failed DD, producing immunologic and clinical benefit in two of two patients before a DD shortage halt. DD alone was well tolerated. Adding IFNα increased toxicities but was tolerable, and reduced human Treg numbers in blood, and function through dendritic cell–induced IL6 in vitro. Conclusions: Treg depletion is clinically useful but unlikely alone to cure ovarian cancer. Rational treatment agent combinations can salvage clinical failure of Treg depletion alone, even when neither single agent provides meaningful clinical benefit.

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., We provide evidence that tumor‐intrinsic PD‐L1 mediates pathologic signals in human and mouse bladder cancers, independent of molecular subtype. Signals include control of proliferation, mTORC1, autophagy, and chemotherapy resistance. We show that countering the pathologic cell‐intrinsic PD‐L1 signals improves chemotherapy response in vivo of mice bearing bladder cancer tumors.

The IL2 receptor (IL2R) is an attractive cancer immunotherapy target that controls immunosuppressive T regulatory cells (Treg) and antitumor T cells. Here we used IL2Rβ-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.

BackgroundAlthough immunotherapy can induce durable anti-tumor response in multiple cancers, immune checkpoint blockade (ICB) therapy resistance in ovarian cancer and melanoma remains problematic. Here, we report that tumor cell-intrinsic mTORC1 regulates ICB response through mTORC1 defining subunit Raptor (Rptor) by modulating interferon-gamma (IFNg) resistance and tumor-initiating cell (TIC) virulence.MethodsWe knocked down two distinct mTORC1 signaling components: Rptor (Rptorlo, aids in mTORC1 assembly) and Lamtor1 (Ltor1lo, docks mTORC1 on lysosomes) in murine ovarian cancer ID8agg and melanoma B16 cells. PD-L1 was CRISPR knocked out in B16 and human ovarian cancer line ES2. Mice with tumors were treated with a-PD-L1± a-CD8 antibody. TICs were estimated by flow-cytometry.1ResultsRptorlo B16 and ID8agg, but not Ltor1lo B16 tumors grew slower and were a-PD-L1 responsive unlike control (ctrl) in WT mice. We noted that ctrl and Rptorlo B16 and ID8agg cells expressed similar surface PD-L1 in vitro. Thus, Rptor suppresses a-PD-L1 response in ICB-resistant tumors. Tumor immune analysis revealed increased CD8+ T cell% and a trend to increased IFNg+CD8+ T cells in a-PD-L1 treated Rptorlo, but not ctrl B16. Rptorlo a-PD-L1 efficacy was lost with a-CD8 and in IFNg knockout mice. In vitro, IFNg suppressed Rptorlo ID8agg proliferation, unlike ctrl. These data suggested that lack of Rptor makes tumors ICB responsive, possibly by making tumors IFNg-sensitive and increasing IFNg+CD8+ T cells. Further, tumor and draining lymph node (DLN) TCF1+PD-1+ T cell stem cells (critical for aPD-L1/PD-1 success2 3) were significantly higher in a-PD-L1 treated Rptorlo tumors. Thus, tumor Rptor status could regulate tumor microenvironment and distal DLN immune landscape on a-PD-L1 treatment.We previously published that mTORC1 promotes PD-L1-dependent tumor proliferation, TIC virulence1 4 PD-L1KO B16 and ES2 cells expressed similar total Rptor protein. However, lower levels of Rptor were loaded in mTOR complex in absence of PD-L1, as assessed by a-mTOR immunoprecipitation, suggesting that pro-tumorigenic Rptor functions were downstream of, and dependent on PD-L1. Successful Rptorlo aPD-L1 treatment reduced TIC in vivo, an effect reversed in absence of CD8+ T cells or host IFNg. Inhibiting ID8agg mTORC1 with rapamycin reduced stemness genes oct4, nanog expression by QPCR. Further, ID8agg Rptorlo TIC formed significantly smaller tumors versus ctrl TIC in immune-compromised NSG mice, confirming their reduced virulence. Rptor, but not Ltor1, expression inversely correlated with tumor CD8+ infiltrate in IMvigor210 trial, and strongly with TIC gene signature in ovarian cancer patients.5 6ConclusionsTumor-cell intrinsic Rptor modulates ICB resistance, IFNg responsiveness, immune microenvironment, and TIC virulence.AcknowledgementsN/ATrial RegistrationN/AReferencesGupta HB, et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. Signal Transduct Target Ther 2016;1,Article number:16030.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Dammeijer F, et al. The PD-1/PD-L1-checkpoint restrains T cell immunity in tumor-draining lymph nodes. Cancer Cell 2020;38:685–700.Clark CA, et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis, and autophagy in ovarian cancer and melanoma. Cancer Res 2016;76:6964–6974.Smith BA, et al. A human adult stem cell signature marks aggressive variants across epithelial cancers. Cell Rep 2018;24:3353–3366.Hoffman-Censits JH, et al. IMvigor 210, a phase II trial of atezolizumab (MPDL3280A) in platinum-treated locally advanced or metastatic urothelial carcinoma (mUC). J Clin Oncol 2016;34, no.2_suppl:355–355.

BackgroundTumor PD-L1 canonically signals to PD-1 on immune cells to evade immune destruction.1 We reported that tumor PD-L1 also mediates diverse pathologic intracellular signals,2 3 including promoting the ATM/Chk2 DNA damage response, and that genetically PD-L1 deficient tumors are sensitized to Chk1 inhibitor therapy.4 DNA damage increases cytosolic DNA, which induces immunogenic STING signals through inflammatory type I interferon and cytokine production.5MethodsWe conducted a high-throughput drug screen that identified the β-lactam cephalosporin antibiotic cefepime as a pharmacologic tumor PD-L1 depleting agent. In vitro tests of β-lactam antibiotics used 80 μM and the Chk1 inhibitors rabusertib and prexasertib were used at indicated concentrations. Cell lines were RT4 human bladder cancer, ID8agg murine ovarian cancer, and B16 mouse melanoma. Viability was by MTT and proteins by immunoblot. We challenged NSG mice (n = 5 per group) with RT4 (SQ) and treated with cefepime (200 mg/kg), rabusertib (2.5 mg/kg), vehicle, or combination daily.ResultsCefepime at pharmacologically relevant concentrations depletes tumor PD-L1 and phenocopies genetic tumor PD-L1 depletion by decreasing Chk2 protein and increasing DNA damage (γH2AX) (figure 1). Chk2 is depleted by cefepime in CTRL cells, but not in PD-L1KO or PD-L1 overexpressing cells, and sensitizes PD-L1+ tumor cells to Chk1 inhibitors in vitro in a PD-L1-dependent manner (figures 1 and 2). Combining cefepime with rabusertib in vivo significantly prolonged severely immunodeficient NSG mice survival in RT4 challenge versus cefepime alone while rabusertib alone was not effective (figure 3). Antimicrobial mechanisms, reported to influence tumor treatment responses,6 are unlikely in NSG mice. To test β-lactam contributions to cefepime efficacy, we found that ceftazidime, a structurally related cephalosporin, also depletes tumor PD-L1 and Chk2 protein and sensitizes tumors to rabusertib in a PD-L1 dependent manner (figures 4 and 5). Structurally-unrelated β-lactam antibiotics did not sensitize tumors to rabusertib. Both cefepime and ceftazidime activate tumor STING, suggesting they could augment immunotherapies by increasing tumor immunogenicity (figure 6).ConclusionsAs genetic PD-L1 depletion is not yet clinically feasible, we provide pharmacologic means to deplete tumor PD-L1 to improve clinical treatment efficacy as a rapidly translated approach. Cefepime is the prototype agent, but ceftazidime is structurally similar with significant activity, suggesting important structure activity relationships to explore. Pharmacologic tumor PD-L1 depletion could augment other standard of care approaches, including immunotherapy, and deserves further investigation.ReferencesDong H, Strome SE, Salomao DR, Tamura H, Fumiya H, Flies DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002;8:793–800.Clark CA, Gupta HB, Gangadhara S, Pandeswara S, Lao S, Yuan B, et al. Tumor-Intrinsic PD-L1 signals regulate cell growth, pathogenesis, and autophagy in Ovarian Cancer and Melanoma. Cancer Res 2016;76(23):6964–6974.3. Gupta HB, Clark CA, Yuan B, Sareddy G, Pandeswara S, Padron AS, et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. Signal Transduct Target Ther 2016;1.Kornepati AV, Zhang D, Hambright HG, Kari SC, Deng Y, Clark CA, et al. Cell-intrinsic programmed death ligand-1 (PD-L1) inhibits cytotoxic chemo, promotes DNA damage repair, and enhances ATM/ATR signaling following exposure to DNA damaging agents in bladder, melanoma, and ovarian cancer cells. J Immunol 2019;202(1):195.17.Gehrke N, Mertens C, Zillinger T, Wenzel J, Bald T, Zahn S, et al. Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing. Immunity 2013;39(3):482–495.Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV, et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science 2018;359(6371):97–103.Ethics ApprovalThis protocol was approved by the UTHSCSA IACUC.Abstract 755 Figure 1Cefepime depletes tumor PD-L1 and Chk2 and induces γH2AXAbstract 755 Figure 2Cefepime sensitizes tumors to Chk1 inhibitors in a PD-L1 dependent mannerAbstract 755 Figure 3Cefepime combined with Chk1 inhibitors in vivo prolongs NSG mouse survivalAbstract 755 Figure 4Ceftazidime depletes tumor PD-L1 and Chk2Abstract 755 Figure 5Ceftazidime, but not other B-lactam antibiotics, sensitizes RT4 to Chk1 inhibitors in a PD-L1 dependent mannerAbstract 755 Figure 6Cefepime and ceftazidime induce STING and p-TBK1

// Neelam Mukherjee 1 , Niannian Ji 1 , Vincent Hurez 2 , Tyler J. Curiel 2 , Maureen O. Montgomery 1 , Andrew J. Braun 1 , Marlo Nicolas 3 , Marcela Aguilera 5 , Dharam Kaushik 1 , Qianqian Liu 4 , Jianhua Ruan 5 , Kerri A. Kendrick 1 and Robert S. Svatek 1 1 Department of Urology, University of Texas Health San Antonio (UTHSA), San Antonio, United States 2 Department of Medicine, University of Texas Health San Antonio (UTHSA), San Antonio, United States 3 Department of Pathology, University of Texas Health San Antonio (UTHSA), San Antonio, United States 4 Department of Epidemiology & Biostatistics, University of Texas Health San Antonio (UTHSA), San Antonio, United States 5 Department of Computer Science, University of Texas San Antonio (UTSA), San Antonio, United States Correspondence to: Robert S. Svatek email: svatek@uthscsa.edu Keywords: bladder cancer; NK cells; tumor-infiltrating lymphocytes; CD56; survival Received: September 26, 2018 Accepted: November 01, 2018 Published: November 23, 2018 ABSTRACT Background: Natural killer (NK) cells are effective at killing tumors in a non-MHC restricted manner and are emerging targets for cancer therapy but their importance in bladder cancer (BC) is poorly defined. NK cells are commonly subdivided into populations based on relative surface expression of CD56. Two major subsets are CD56 bright and CD56 dim NK cells. Methods: The prevalence of intratumoral lymphocytes was examined via flow cytometric analysis of bladder tissue from a local cohort of patients with non-invasive and invasive BC (n=28). The association of NK cell subsets with cancer-specific survival (CSS) and overall survival (OS) was examined in 50 patients with BC using Cox regression. Fluorescence-activated cell sorting (FACS) of intratumoral lymphocytes isolated CD56 NK cell subsets were used for examination of function, including cytokine production and in vitro cytotoxicity. Results: NK cells predominated among bladder intratumoral lymphocytes. Intratumoral CD56 bright NK cells showed increased cytokine production and cytotoxicity compared to their CD56 dim counterparts and were associated with improved CSS and OS independent of pathologic tumor stage. On the other hand, CD56 dim NK cells were not associated with improved outcomes but were associated with higher pathologic stage. Conclusions: NK cells are frequent among intratumoral lymphocytes in BC. Bladder intratumoral CD56 bright NK cells are functional and prognostically relevant whereas CD56 dim NK cells are dysfunctional and prevalent in higher stage tumors. Thus, CD56 bright NK cells are promising targets in BC.

BackgroundAlthough intravesical BCG is the standard treatment of high-grade non-muscle invasive bladder cancer (NMIBC), response rates remain unsatisfactory. In preclinical models, rapamycin enhances BCG vaccine efficacy against tuberculosis and the killing capacity of γδ T cells, which are critical for BCG’s antitumor effects. Here, we monitored immunity, safety, and tolerability of rapamycin combined with BCG in patients with NMIBC.MethodsA randomized double-blind trial of oral rapamycin (0.5 or 2.0 mg daily) versus placebo for 1 month was conducted in patients with NMIBC concurrently receiving 3 weekly BCG instillations (NCT02753309). The primary outcome was induction of BCG-specific γδ T cells, measured as a percentage change from baseline. Post-BCG urinary cytokines and immune cells were examined as surrogates for local immune response in the bladder. Secondary outcomes measured were adverse events (AEs) and tolerability using validated patient-reported questionnaires.ResultsThirty-one patients were randomized (11 placebo, 8 rapamycin 2.0 mg, and 12 rapamycin 0.5 mg). AEs were similar across groups and most were grade 1–2. One (12.5%) patient randomized to 2.0 mg rapamycin was taken off treatment due to stomatitis. No significant differences in urinary symptoms, bowel function, or bother were observed between groups. The median (IQR) percentage change in BCG-specific γδ T cells from baseline per group was as follows: −26% (−51% to 24%) for placebo, 9.6% (−59% to 117%) for rapamycin 0.5 mg (versus placebo, p=0.18), and 78.8% (−31% to 115%) for rapamycin 2.0 mg (versus placebo, p=0.03). BCG-induced cytokines showed a progressive increase in IL-8 (p=0.02) and TNF-α (p=0.04) over time for patients on rapamycin 2.0 mg, whereas patients receiving placebo had no significant change in urinary cytokines. Compared with placebo, patients receiving 2.0 mg rapamycin had increased urinary γδ T cells at the first week of BCG (p=0.02).ConclusionsFour weeks of 0.5 and 2.0 mg oral rapamycin daily is safe and tolerable in combination with BCG for patients with NMIBC. Rapamycin enhances BCG-specific γδ T cell immunity and boosts urinary cytokines during BCG treatment. Further study is needed to determine long-term rapamycin safety, tolerability and effects on BCG efficacy.

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.

Background Aging is the biggest risk factor for cancer, yet little is known about cancer immunotherapy effects. ?PD-1 can block PD-L1 and PD-L2 while ?PD-L1 blocks PD-1 and CD80.1 A recent key finding in young hosts including humans is that melanoma response to aPD-1/aPD-L1 correlates with CD8+TCF-1+ T cell stem cell (TCSC) generation.2 Methods We tested aPD-1 (100 or 200 µg/mouse), aPD-L1 (100 µg/mouse) or aPD-L2 (200 µg/mouse) in aged (18-33 months) and young (3-8 months) mice challenged orthotopically with B16 (WT or PD-L1ko) melanoma (SQ) or ID8agg ovarian cancer (IP). Tumors were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF. Results We reported that aPD-1 treats young and aged with B16 and aPD-L1 treats young not aged.3 aPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibiting this property [figure 1]. Efficacy in young (aPD-1, aPD-L1) and aged (aPD-L2) correlated with increased tumor TCSC [figure 3], but TCSC differed by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3) [figure 2]. aPD-L2 efficacy against B16 in aged mice required host IFN-g and IL-17 [figure 4]. IP ID8agg ovarian cancer did not respond to aPD-L2 in aged or young mice. Aged expressed up to 40-fold more PD-L2 versus young on various immune cells suggesting high PD-L2 helps aPD-L2 response [figure 6]. Host IFN-g contributed to aged PD-L2 expression, which did not appear cell-autonomous [figure 6]. PD-L1KO aged but not young mice challenged with PD-L1KO B16 responded to aPD-1 [figure 5], consistent with PD-L2 block as a mechanism. Conclusions Treatment differences in aged versus young could depend on immune checkpoint or TCSC differences, which could be related to CD8+ T-cell infiltration, including TCSC. aPD-1 efficacy in aged PD-L1KO mice challenged with PD-L1KO B16 suggests that aPD-1 efficacy is through PD-L2 block in aged. PD-L2 expression differences and anatomical compartment differences in tumor microenvironment may also contribute to treatment efficacy differences. We are now identifying mechanisms for increased PD-L2 and other mechanisms for aPD-L2 efficacy in aged, and testing TCSC effects. Our work can improve cancer immunotherapy in aged hosts and provides insights in treatment failures, including in young hosts. Acknowledgements South Texas MSTP training grant (NIH T32GM113896), TL1TR002647, Graduate Research in Immunology Program training grant (NIH T32 AI138944), R01 CA231325, Samuel Waxman Cancer Research Foundation Grant Ethics Approval The study was approved by UTHSA IACUC, approval number 20180021. References Schildberg FA, Klein SR, Freeman GJ, Sharpe AH. Coinhibitory pathways in the B7-CD28 ligand-receptor family. Immunity 2016;44(5):955-72. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537(7620):417-21. Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol 2018;105:146-54.

The programmed death-ligand 1 (PD-L1)-dependent immune checkpoint attenuates host immunity and maintains self-tolerance. Imbalance between protective immunity and immunopathology due to altered PD-L1 signaling can lead to autoimmunity or tumor immunosuppression. The role of the PD-L1-dependent checkpoint in non-immune system is less reported. We previously found that white adipocytes highly express PD-L1. Here we show that adipocyte-specific PD-L1 knockout mice exhibit enhanced host anti-tumor immunity against mammary tumors and melanoma with low or no tumor PD-L1. However, adipocyte PD-L1 ablation in tumor-free mice also exacerbates diet-induced body weight gain, pro-inflammatory macrophage infiltration into adipose tissue, and insulin resistance. Low PD-L1 mRNA levels in human adipose tissue correlate with high body mass index and presence of type 2 diabetes. Therefore, our mouse genetic approach unequivocally demonstrates a cell-autonomous function of adipocyte PD-L1 in promoting tumor growth and inhibiting antitumor immunity. In addition, our work uncovers a previously unrecognized role of adipocyte PD-L1 in mitigating obesity-related inflammation and metabolic dysfunction.

Regulatory T cells (Treg) are critical mediators of immunosuppression in established tumors, although little is known about their role in restraining immunosurveillance during tumorigenesis. Here, we employ an inducible autochthonous model of melanoma to investigate the earliest Treg and CD8 effector T-cell responses during oncogene-driven tumorigenesis. Induction of oncogenic BRAFV600E and loss of Pten in melanocytes led to localized accumulation of FoxP3+ Tregs, but not CD8 T cells, within 1 week of detectable increases in melanocyte differentiation antigen expression. Melanoma tumorigenesis elicited early expansion of shared tumor/self-antigen–specific, thymically derived Tregs in draining lymph nodes, and induced their subsequent recruitment to sites of tumorigenesis in the skin. Lymph node egress of tumor-activated Tregs was required for their C-C chemokine receptor 4 (Ccr4)–dependent homing to nascent tumor sites. Notably, BRAFV600E signaling controlled expression of Ccr4-cognate chemokines and governed recruitment of Tregs to tumor-induced skin sites. BRAFV600E expression alone in melanocytes resulted in nevus formation and associated Treg recruitment, indicating that BRAFV600E signaling is sufficient to recruit Tregs. Treg depletion liberated immunosurveillance, evidenced by CD8 T-cell responses against the tumor/self-antigen gp100, which was concurrent with the formation of microscopic neoplasia. These studies establish a novel role for BRAFV600E as a tumor cell–intrinsic mediator of immune evasion and underscore the critical early role of Treg-mediated suppression during autochthonous tumorigenesis. Significance: This work provides new insights into the mechanisms by which oncogenic pathways impact immune regulation in the nascent tumor microenvironment. Cancer Res; 78(17); 5038–49. ©2018 AACR.

Programmed death-ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) play important roles in modulating antitumor immune response and are targeted by checkpoint blockade immunotherapy. While PD-L1 expression in both tumor and host cells is associated with antitumor therapeutic efficacy, the exact contribution of PD-L1 in various tissue and cell compartments to antitumor immune response remains to be elucidated. Here we show that PD-L1 expression is markedly elevated in human and mouse mature adipocytes compared to their preadipocyte counterparts. When co-cultured with mouse splenocytes in vitro, adipocytes prevent anti-PD-L1 antibody from activating CD8+T cells. Genetic ablation of adipose PD-L1 obliterates the inhibitory effect of adipocytes on anti-PD-L1 antibody. Conversely, enforced PD-L1 expression in preadipocytes confers the antibody-inhibitory activity. GW9662, a pharmacologic inhibitor of peroxisome proliferator-activated receptor γ (PPARγ) in adipogenesis, selectively reduces PD-L1 expression in mouse adipose tissue. The same PPARγ antagonist also enhances the antitumor efficacy of checkpoint blockade antibodies for treating multiple mammary tumors. Our findings provide a previously unappreciated approach to bolster anticancer immunotherapy efficacy and suggest a mechanism for the role of adipose tissue in breast cancer progression. Citation Format: Wu B, Sun X, Gupta HB, Yuan B, Ge F, Li J, Hu Y, Curiel TJ, Li R. Adipose PD-L1 modulates checkpoint blockade immunotherapy efficacy in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-30.

BackgroundTumor surface-expressed programmed death-ligand 1 (PD-L1) suppresses immunity when it engages programmed death-1 (PD-1) on anti-tumor immune cells in canonical PD-L1/PD-1.1 Non-canonical, tumour-intrinsic PD-L1 signals can mediate treatment resistance2–6 but mechanisms remain incompletely understood. Targeting non-canonical, cell-intrinsic PD-L1 signals, especially modulation of the DNA damage response (DDR), remains largely untapped.MethodsWe made PD-L1 knockout (PD-L1 KO) murine transplantable and human cell lines representing melanoma, bladder, and breast histologies. We used biochemical, genetic, and cell-biology techniques for mechanistic insights into tumor-intrinsic PD-L1 control of specific DDR and DNA repair pathways. We generated a novel inducible melanoma GEMM lacking PD-L1 only in melanocytes to corroborate DDR alterations observed in PD-L1 KO of established tumors.ResultsGenetic tumor PD-L1 depletion destabilized Chk2 and impaired ATM/Chk2, but not ATR/Chk1 DDR. PD-L1KO increased DNA damage (γH2AX) and impaired homologous recombination DNA repair (p-RPA32, BRCA1, RAD51 nuclear foci) and function (DR-GFP reporter). PD-L1 KO cells were significantly more sensitive versus controls to DDR inhibitors (DDRi) against ATR, Chk1, and PARP but not ATM in multiple human and mouse tumor models in vitro and in vivo in NSG mice. PD-1 independent, intracellular, not surface PD-L1 stabilized Chk2 protein with minimal Chek2 mRNA effect. Mechanistically, PD-L1 could directly complex with Chk2, protecting it from PIRH2-mediated polyubiquitination. PD-L1 N-terminal domains Ig-V and Ig-C but not the PD-L1 C-terminal tail co-IP’d with Chk2 and restored Chk1 inhibitor (Chk1i) treatment resistance. Tumor PD-L1 expression correlated with Chk1i sensitivity in 44 primary human small cell lung cancer cell lines, implicating tumor-intrinsic PD-L1 as a DDRi response biomarker. In WT mice, genetic PD-L1 depletion but not surface PD-L1 blockade with αPD-L1, sensitized immunotherapy-resistant, BRCA1-WT 4T1 tumors to PARP inhibitor (PARPi). PARPi effects were reduced on PD-L1 KO tumors in RAG2KO mice indicating immune-dependent DDRi efficacy. Tumor PD-L1 depletion, likely due to impaired DDR, enhanced PARPi induced tumor-intrinsic STING activation (e.g., p-TBK1, CCL5) suggesting potential to augment immunotherapies.ConclusionsWe challenge the prevailing surface PD-L1 paradigm and establish a novel mechanism for cell-intrinsic PD-L1 control of the DDR and gene product expression. We identify therapeutic vulnerabilities from tumor PD-L1 depletion utilizing small molecule DDRi currently being tested in clinical trials. Data could explain αPD-L1/DDRi treatment resistance. Intracellular PD-L1 could be a pharmacologically targetable treatment target and/or response biomarker for selective DDRi alone plus other immunotherapies.ReferencesTopalian SL, Taube JM, Anders RA, Pardoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer 16:275–287, doi:10.1038/nrc.2016.36 (2016).Clark CA, et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis and autophagy in ovarian cancer and melanoma. Canres 0258.2016 (2016).Gupta HB et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. 1, 16030 (2016).Zhu H, et al. BET bromodomain inhibition promotes anti-tumor immunity by suppressing PD-L1 expression. Cell Rep 16:2829–2837, doi:10.1016/j.celrep.2016.08.032 (2016)Wu B, et al. Adipose PD-L1 modulates PD-1/PD-L1 checkpoint blockade immunotherapy efficacy in breast cancer. Oncoimmunology 7:e1500107, doi:10.1080/2162402X.2018.1500107 (2018)Liang J, et al. Verteporfin inhibits PD-L1 through autophagy and the STAT1-IRF1-TRIM28 signaling axis, exerting antitumor efficacy. Cancer Immunol Res 8:952–965, doi:10.1158/2326-6066.CIR-19-0159 (2020)

BackgroundProgrammed cell death ligand-1 (PD-L1) overexpression in tumor cells inhibits T cells activity and delivers pathologic intracellular signals that can reduce cancer treatment responses in pre-clinicalmodels.1 2MethodsTo reduce tumor intracellular PD-L1-mediated pathology, we performed a drug screen that identified chlorambucil as a tumor cell PD-L1 depletion drug.ResultsChlorambucil depletes basal tumor PD-L1 expression through the ubiquitination proteasome pathway. In the tumor microenvironment, high chlorambucil doses treated orthotopic B16 melanoma and ID8agg ovarian cancer. Chlorambucil treatment efficacy was lost or reduced in PD-L1lo ID8agg and PD-L1KO B16 tumors, corroborated with in vitro data. These data suggest that chlorambucil anti-tumor activity of CAMB requires tumor PD-L1 expression, confirmed in PD-L1KO host challenge with CTRL tumor, which chlorambucil treated effectively. Chlorambucil rendered αPD-L1 resistant CTRL ID8agg and PD-L1lo B16 tumors αPD-L1 sensitive, preliminarily possibly due to tumor STING activation, and associated with enhanced tumor NK cell infiltration and central memory T cell generation. Chlorambucil also phenocopied genetic PD-L1KO by reducing tumor cell mTORC1 signals and stem cell content,3 suggesting additional treatment potential.ConclusionsChlorambucil could be a useful strategy to reprogram tumor PD-L1 signals and boost immune-based therapies especially for anti-PD-L1-resistant tumors.ReferencesClark CA et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis, and autophagy in ovarian cancer and melanoma. Cancer Res 2016;76:6964–6974.Juneja VR et al. PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med 2017;214:895–904.Gupta HB et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. Signal Transduct Target Ther 2016;1:2095–9907.Ethics ApprovalWe received approval from the UT Health San Antonio Institutional Animal Care and Use Committee (IACUC) for each procedure that used mice. We conducted each experiment per the standards required by the UT Health San Antonio Department of Laboratory Animal Resources.

B cells promote tumor development in murine models of breast cancer (BCa), including - as we have shown - by producing pleiotropic cytokine IL-27, which upregulates expression of the PD-L1 immune checkpoint in BCa cells and tumor-infiltrating B cells (TIL-Bs). Paradoxically, B cell activation has been shown to predict the positive response to immune checkpoint inhibitors (ICIs) by murine BCa tumors and melanoma, sarcoma and kidney cancer in patients. Here, we have addressed the role of B cell-expressed PD-L1 in BCa development and responses to ICIs by generating conditional knockout (KO) mice in which Cd274 (encoding PD-L1) was ablated only in activated B cells (AicdacreCd274fl/fl). Tumor development in AicdacreCd274fl/fl mice from engrafted syngeneic ER+ E0071 BCa cells was significantly delayed, albeit not abolished (as occurring in mice lacking B cells), in association with increased B-cell expression of CD86, a key agonistic ligand for T cell-expressed CD28, a co-stimulatory receptor whose function is inhibited by the PD-L1/PD-1 immune checkpoint in exhausted tumor-infiltrating CD8+ T cells. The heightened B-cell CD86 expression in AicdacreCd274fl/fl mice was recapitulated in AicdacreCd274fl/fl B cells stimulated in vitro by CD154, a T helper cell stimulus critical for B cell activation and differentiation as well as the antibody response. Accordingly, AicdacreCd274fl/fl mice displayed an increased antibody response to a T-dependent antigen. Likewise, treatment with the anti-PD-L1 (anti-PD-L1) ICI boosted wildtype B cells stimulated with CD154 in vitro to express CD86 and differentiate into potentiated plasma cells. It also effectively regressed the growth of pre-developed E0071 tumor in C57 and AicdacreCd274+/fl mice by rejuvenating tumor-infiltrating CD8+ T cells, likely through activation of previously dampened CD28 signaling. By contrast, residual tumors that had eventually developed in AicdacreCd274fl/fl mice failed to respond to αPD-L1 and continued to grow at a pace comparable to tumors treated with an isotype-matched control antibody, showing that the full anti-tumor efficacy of anti-PD-L1 was mediated mainly by PD-L1-expressing B cells, but not other immune cell types. Thus, PD-L1+ TIL-Bs use PD-L1 to impose a “brake” on the host anti-tumor activity, but are directly targeted by αPD-L1 to relieve the brake, “jump-start” the CD86:CD28 signaling and potentiate the anti-tumor response, thereby providing a mechanistic explanation for the opposing roles of B cells in murine BCa development and ICI responses. Our findings also provide a strong rationale to investigate the activation of PD-L1-expressing TIL-Bs by Atezolizumab (anti-PD-L1) in BCa patients to underpin the patient response to this ICI. Citation Format: Mustafa T. Khan, Christian Cervantes, Zexuan Liu, Suryavathi Viswanadhapalli, Harshita Gupta, Tyler Curiel, Ratna Vadlamudi, Zhenming Xu. PD-L1-expressing B cells promote murine breast cancer development and mediate the response to anti-PD-L1 immune checkpoint inhibitor to upregulate B-cell CD86 and inhibit tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB160.

Ovarian cancer is the deadliest of all gynecologic cancers. Despite success with initial chemotherapy, the majority of patients relapse with an incurable disease. Development of chemotherapy resistance is a major factor for poor long-term survival in ovarian cancer. The biological effects of estrogens are mediated by estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Emerging evidence suggests that ovarian cancer cells express ERβ that functions as a tumor suppressor; however, the clinical utility of ERβ agonists in ovarian cancer remains elusive. We tested the utility of two natural ERβ agonists liquiritigenin (Liq), which is isolated from Glycyrrhiza uralensis and S-equol, which is isolated from soy isoflavone daidzein, for treating ovarian cancer. Both natural ERβ ligands had significant growth inhibition in cell viability and survival assays, reduced migration and invasion, and promoted apoptosis. Further, ERβ agonists showed tumor suppressive functions in therapy-resistant ovarian cancer model cells and sensitized ovarian cancer cells to cisplatin and paclitaxel treatment. Global RNA-Seq analysis revealed that ERβ agonists modulate several tumor suppressive pathways, including downregulation of the NF-κB pathway. Immunoprecipitation assays revealed that ERβ interacts with p65 subunit of NF-κB and ERβ overexpression reduced the expression of NF-κB target genes. In xenograft assays, ERβ agonists reduced tumor growth and promoted apoptosis. Collectively, our findings demonstrated that natural ERβ agonists have the potential to significantly inhibit ovarian cancer cell growth by anti-inflammatory and pro-apoptotic actions, and natural ERβ agonists represent novel therapeutic agents for the management of ovarian cancer.

mTOR drives tumor growth but also supports T-cell function, rendering the applications of mTOR inhibitors complex especially in T-cell malignancies. Here, we studied the effects of the mTOR inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical pharmacologic rapamycin (1–8 mg/kg) significantly reduced tumor burden via direct suppression of tumor cell proliferation and improved survival in EL4 challenge independent of antitumor immunity. Denileukin diftitox (DD)–mediated depletion of regulatory T cells significantly slowed EL4 growth in vivo in a T-cell–dependent fashion. However, typical rapamycin inhibited T-cell activation and tumor infiltration in vivo and failed to boost DD treatment effects. Low-dose (LD) rapamycin (75 μg/kg) increased potentially beneficial CD44hiCD62L+ CD8+ central memory T cells in EL4 challenge, but without clinical benefit. LD rapamycin significantly enhanced DD treatment efficacy, but DD plus LD rapamycin treatment effects were independent of antitumor immunity. Instead, rapamycin upregulated EL4 IL2 receptor in vitro and in vivo, facilitating direct DD tumor cell killing. LD rapamycin augmented DD efficacy against B16 melanoma and a human B-cell lymphoma, but not against human Jurkat T-cell lymphoma or ID8agg ovarian cancer cells. Treatment effects correlated with IL2R expression, but mechanisms in some tumors were not fully defined. Overall, our data define a distinct, biphasic mechanisms of action of mTOR inhibition at doses that are clinically exploitable, including in T-cell lymphomas. Cancer Res; 77(2); 520–31. ©2016 AACR.

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.

Programmed death-ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) modulate antitumor immunity and are major targets of checkpoint blockade immunotherapy. However, clinical trials of anti-PD-L1 and anti-PD-1 antibodies in breast cancer demonstrate only modest efficacy. Furthermore, specific PD-L1 contributions in various tissue and cell compartments to antitumor immunity remain incompletely elucidated. Here we show that PD-L1 expression is markedly elevated in mature adipocytes versus preadipocytes. Adipocyte PD-L1 prevents anti-PD-L1 antibody from activating important antitumor functions of CD8+ T cells in vitro. Adipocyte PD-L1 ablation obliterates, whereas forced preadipocyte PD-L1 expression confers, these inhibitory effects. Pharmacologic inhibition of adipogenesis selectively reduces PD-L1 expression in mouse adipose tissue and enhances the antitumor efficacy of anti-PD-L1 or anti-PD-1 antibodies in syngeneic mammary tumor models. Our findings provide a previously unappreciated approach to bolster anticancer immunotherapy efficacy and suggest a mechanism for the role of adipose tissue in breast cancer progression.

BackgroundAnti-programmed death-ligand 1 (αPD-L1) immunotherapy is approved to treat bladder cancer (BC) but is effective in + 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.MethodsWe 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.ResultsIL-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.ConclusionsMechanistic 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.

BackgroundThe non-overlapping functions of the two estrogen receptor subtypes, ERα (Estrogen Receptor α)and ERβ (Estrogen Receptor β), in tumor cells have been studied extensively. However, their counterparts in host cells is vastly underinterrogated. Even less is known about how ERα and ERβ activities are regulated in a subtype-specific manner. We previously identified a phosphotyrosine residue (pY36) of human ERβ that is important for tumor ERβ to inhibit growth of breast cancer cells in vitro and in vivo. A role of this ERβ phosphotyrosine switch in regulating host ERβ remains unclear.Conventional gene editing was used to mutate the corresponding tyrosine residue of endogenous mouse ERβ (Y55F) in mouse embryonic stem cells. The derived homozygous mutant Esr2Y55F/Y55F mouse strain and its wild-type (WT) counterpart were compared in various transplant tumor models for their ability to support tumor growth. In addition, flow cytometry-based immunophenotyping was carried out to assess antitumor immunity of WT and mutant hosts. Adoptive transfer of bone marrow and purified CD8+ T cells were performed to identify the host cell type that harbors ERβ-dependent antitumor function. Furthermore, cell signaling assays were conducted to compare T cell receptor (TCR)-initiated signaling cascade in CD8+ T cells of WT and mutant mice. Lastly, the ERβ-selective agonist S-equol was evaluated for its efficacy to boost immune checkpoint blockade (ICB)-based anticancer immunotherapy.Disabling the ERβ-specific phosphotyrosine switch in tumor-bearing hosts exacerbates tumor growth. Further, a cell-autonomous ERβ function was defined in CD8+ effector T cells. Mechanistically, TCR activation triggers ERβ phosphorylation, which in turn augments the downstream TCR signaling cascade via a non-genomic action of ERβ. S-equol facilitates TCR activation that stimulates the ERβ phosphotyrosine switch and boosts anti-PD-1 (Programmed cell death protein 1) ICB immunotherapy.Our mouse genetic study clearly demonstrates a role of the ERβ phosphotyrosine switch in regulating ERβ-dependent antitumor immunity in CD8+ T cells. Our findings support the development of ERβ agonists including S-equol in combination with ICB immunotherapy for cancer treatment.

Unlike estrogen receptor α (ERα) that predominantly promotes hormone-dependent breast tumor growth, ERβ exhibits antitumor effects in a variety of cancer types. We recently identified a phosphotyrosine residue in ERβ, but not ERα, that dictates ERβ transcriptional activity and antitumor function. We show here that this ER isotype-specific phosphotyrosine switch is important for regulating ERβ activity in cell proliferation, migration, and invasion. At the mechanistic level, phosphorylated ERβ, which recruits transcriptional coactivator p300, is in turn targeted by p300 for ubiquitination and proteasome-dependent protein turnover. Furthermore, ERβ-specific agonists such as S-equol enhance ERβ phosphorylation, suggesting a crosstalk between ligand- and posttranslational modification-dependent ERβ activation. Inhibition of xenograft tumor growth by S-equol is associated with reduced tumor Ki-67 expression and elevated ERβ tyrosine phosphorylation. Taken together, our data support the notion that phosphotyrosine-dependent ERβ signaling is an attractive target for anticancer treatment.

We sought to determine the subcellular distribution of 30+ immune-related markers including PD-L1 in stage III melanoma tumor tissues (nuclear, cytoplasmic, surface) using novel imaging mass cytometry technology that enables simultaneous detection of over 30 immune and tumor markers. Metal-tagged antibodies were used to stain stage III melanoma tissues similarly to an IHC-based protocol, followed by laser ablation and mass cytometry using the Helios® and Hyperion® technology (Fluidigm). Markers for immune subsets included CD45, CD3, CD4, CD8, PD-1, Granzyme B, FoxP3, CD11b, CD11c, CD107, CD20, Vista, and CD68. Markers for tumor cell phenotyping included PD-L1 (intracellular and extracellular domain specific), E-cadherin, B-catenin, PD-L2, and keratin 8/18. Structural, inflammatory, survival, and signaling markers included Ki67, p53, p-tyrosine, Bcl-2, Cl-caspase 3, histone H3, collagen-1, actin, and arginase-1. We further validated subcellular distribution of PD-L1 in the same patient tumor cores using fluorochrome-labeled antibodies and 100X confocal microscopy to show difference in surface-only, cytoplasmic-only, and nuclear PD-L1. We then assessed the association between subcellular PD-L1 and immune populations in the tumor microenvironment, along with overall and recurrence-free survival. We wrote custom algorithms for data processing to identify novel immune subsets, ran SPADE and viSNE analyses for cross-validation, and employed Imaris Bitplane software to validate our own algorithms for immune population identification. Overall, we found that intracellular-only PD-L1 in tumor tissues has the highest correlation (Pearson r) to CD11b, FoxP3, PD-1, arginase, Ki67, PD-L2 and nuclear stain iridium, while extracellular (surface) PD-L1 in tumors has the highest correlation (Pearson r) to FoxP3, PD-1, arginase, Ki67, PD-L2 and Vista. We identified two novel immunophenotypes (histone-3+/p-tyrosinehi /Vistalo / PD-L1-ICDlo / PD-L1-ECDlo and CD11bhi / PD-L1-ICDhi / PD-L1-ECDhi) that are positively associated with overall patient survival. Of note, only phospho-Tyrhi was significantly associated with RFS. This work highlights the use of cutting-edge technology that can employ the highest multiplexing available for assessing novel interactions of 30+ tumor tissue markers for identification of novel immunophenotypes and biomarker in stage III melanoma patients. Our novel study showcases a novel methodologic approach to high-throughput analysis of retrospective profiling of the melanoma tumor immune microenvironment on a single IHC slide, followed by appropriate and rigorous cross-validation using Fluidigm’s novel CyTOF imaging technology. Our study provides a groundwork for this powerful, novel technology in translational and clinical research for biomarker identification and identification of novel combinatorial markers that may predict overall and recurrence-free survival in stage III melanoma. Citation Format: Heather G. Hambright, Anand V.R. Kornepati, Dai Ogata, Roland R.L. Bassett, Suhendan Ekmekcioglu, Elizabeth A. Grimm, Tyler J. Curiel. High-dimensional (30-plex) imaging mass cytometry on tissue microarray identifies novel PD-L1-inclusive immunophenotypes associated with overall survival in stage III melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B10.

B cells frequently infiltrate breast cancer (BCa) tumors, but their roles are inadequately understood. As shown by our analyses of the TCGA datasets and human BCa arrays, tumoral expression of B cell hallmark factors (e.g., CD20 and PAX5) coincides with those of the two subunits of pleiotropic cytokine IL-27, EBI3 and IL-27p28, whose expression is associated with poor prognosis in patients. In syngeneic mouse models, B cells promote BCa progression and do so by producing IL-27, but not IL-35 (IL-12/EBI3 heterodimer). Upon treatment by recombinant IL-27 or co-culturing with IL-27-producing B cells, BCa cells as well as targeted B lymphocytesactivate STAT1/STAT3 and upregulate immune checkpoint PD-L1 and chemokine receptor CXCR3, blocking of which synergistically arrest BCa cell proliferation in vitro and tumor growth in vivo, concomitant with reduction in PD-L1+B cells. Finally, B cell-specific deficiency in IL-27 receptor or PD-L1 inhibits BCa tumorigenesis, showing the dual functions of tumor-infiltrating B cells, i.e., IL-27 production for paracrinic PD-L1 upregulation. Citation Format: Hui Yan, Suryavathi Viswanadhapalli, Dariela Perez, Daniel Chupp, Maria Fernandez, Jingwei Wang, Shuai Wu, Carlos E. Rivera, Justin B. Moroney, Julia Taylor, John Im, Yiliao Luo, Junhao Liu, Sareddy Gangadhara, Tyler Curiel, Paolo Casali, Ratna Vadlamudi, Zhenming Xu. B cells produce IL-27 in breast cancer to upregulate PD-L1 expression and promote tumor progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2654.

The host immune response can be evaded by cancer cells, e.g., through upregulation of the PD-L1/PD-1 immune checkpoint, and even hijacked to promote tumor progression. Here, we show that tumor-infiltrating B lymphocytes play an important role in PD-L1 expression using two different murine models of breast cancer (BCa). Mice deficient in B cells displayed virtually abrogatedtumor growth from transplanted syngeneic BCa cells and expressed less PD-L1 in residual tumors. Mechanistic studies showed that the impact of B cells was through their production of IL-27, a pleotropic cytokine composed of the IL-27p28/EBI3 heterodimer and possessing both pro- and anti-inflammatory functions. Accordingly, mice with B cell-specific deficiency in expressing IL-27p28/EBI3(but not Il12a, which can heterodimerize with EBI3 to form cytokine IL-35) were severely impaired in the BCa tumor growth and tumoral PD-L1 expression. IL-27-producing B cells were generated by priming of B cells with TLR ligands (that are abundant in the tumor microenvironment due to the release by apoptotic cells) followed by stimulation with CD154 and IL-21 (as likely expressed by activated tumor-infiltrating CD4+T cells). Single cell-RNA-sequencing analysis using Il27p28-Gfp reporter mice revealed that IL-27-producing B cells expressed a transcriptome that is distinct than other B cells and IL-27-producing non-B cells. B cell secreted IL-27, induced STAT1 and STAT3 activation, upregulated PD-L1 expression, and enhanced the growth of human and mouse BCa cells in vitro.It also activated STAT1/STAT3 and upregulated PD-L1 in B lymphocytes, which were the immune cell type with the highest PD-L1 expression in murine BCa tumors. In human primaryBCa tumors, stronger IL-27 expression was associatedwith more B cell infiltration and PD-L1 expression, and correlated with a poorer survival in BCa patients. Further, recombinant IL-27 supported human BCa tumor growth in mice with humanized immune system. Collectively, these results demonstrated that B cell production of IL-27 underpins how the host immune system is hijacked to promote BCa tumor progression. Supported by DOD/BCRP grant BC170448 (to R.V. and Z.X.), NIH/NIAID grant AI 135599 and UT Health Mays Cancer Center - Dew Foundation Pilot Grant (to Z.X.). Citation Format: Hui Yan, Suryavathi Viswanadhapalli, Daniel Chupp, Maria Fernandez, Shuai Wu, Jingwei Wang, Justin Moroney, Julia Taylor, John Im, Carlos Rivera, Yiliao Luo, Junhao Liu, Gangadhara Sareddy, Tyler Curiel, Paolo Casali, Ratna Vadlamudi, Zhenming Xu. Tumor-infiltrating B lymphocytes produce IL-27 to upregulate PD-L1 expression in the tumor microenvironment and promote breast cancer progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-05.

Ovarian cancer (OC) is an immunogenic tumor and among the first where measures of anti-tumor immunity correlated with improved survival. Thus, immunotherapy could be a viable OC treatment modality. Nonetheless, clinical OC immunotherapy trials have demonstrated only modest successes at best, and there is currently no Food and Drug Administration (FDA)approved OC immune therapy despite recent successes in other carcinomas and lymphomas and FDA-approved immunotherapy agents for them. New data suggest specific impediments to effective de novo and treatment-induced anti-OC immunity and support the concept that effective, tolerable OC immunotherapy could be developed based on these newer insights. This chapter reviews the history of OC immunotherapy, highlights important discoveries in OC-related immune dysfunction, covers promising recent developments, highlights newer and ongoing clinical trials, and speculates on future directions that could lead to improved OC immunotherapy approaches.

Cancer prevention is a cost-effective alternative to treatment. In mice, the mTOR inhibitor rapamycin prevents distinct spontaneous, noninflammatory cancers, making it a candidate broad-spectrum cancer prevention agent. We now show that oral microencapsulated rapamycin (eRapa) prevents skin cancer in dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) carcinogen-induced, inflammation-driven carcinogenesis. eRapa given before DMBA/TPA exposure significantly increased tumor latency, reduced papilloma prevalence and numbers, and completely inhibited malignant degeneration into squamous cell carcinoma. Rapamycin is primarily an mTORC1-specific inhibitor, but eRapa did not reduce mTORC1 signaling in skin or papillomas, and did not reduce important proinflammatory factors in this model, including p-Stat3, IL17A, IL23, IL12, IL1β, IL6, or TNFα. In support of lack of mTORC1 inhibition, eRapa did not reduce numbers or proliferation of CD45−CD34+CD49fmid skin cancer initiating stem cells in vivo and marginally reduced epidermal hyperplasia. Interestingly, eRapa reduced DMBA/TPA-induced skin DNA damage and the hras codon 61 mutation that specifically drives carcinogenesis in this model, suggesting reduction of DNA damage as a cancer prevention mechanism. In support, cancer prevention and DNA damage reduction effects were lost when eRapa was given after DMBA-induced DNA damage in vivo. eRapa afforded picomolar concentrations of rapamycin in skin of DMBA/TPA-exposed mice, concentrations that also reduced DMBA-induced DNA damage in mouse and human fibroblasts in vitro. Thus, we have identified DNA damage reduction as a novel mechanism by which rapamycin can prevent cancer, which could lay the foundation for its use as a cancer prevention agent in selected human populations. Cancer Prev Res; 8(5); 400–9. ©2015 AACR.

miR155 is a regulator of immune cell development and function that is generally thought to be immunostimulatory. However, we report here that genetic ablation of miR155 renders mice resistant to chemical carcinogenesis and the growth of several transplanted tumors, suggesting that miR155 functions in immunosuppression and tumor promotion. Host miR155 deficiency promoted overall antitumor immunity despite the finding of defective responses of miR155-deficient dendritic cells and antitumor T cells. Further analysis of immune cell compartments revealed that miR155 regulated the accumulation of functional myeloid-derived suppressive cells (MDSC) in the tumor microenvironment. Specifically, miR155 mediated MDSC suppressor activity through at least two mechanisms, including SOCS1 repression and a reduced ability to license the generation of CD4+Foxp3+ regulatory T cells. Importantly, we demonstrated that miR155 expression was required for MDSC to facilitate tumor growth. Thus, our results revealed a contextual function for miR155 in antitumor immunity, with a role in MDSC support that appears to dominate in tumor-bearing hosts. Overall, the balance of these cellular effects appears to be a root determinant of whether miR155 promotes or inhibits tumor growth. Cancer Res; 75(3); 519–31. ©2014 AACR.

Purpose: To determine the safety and toxicities of sequential MMC (mitomycin C) + BCG (bacillus Calmette–Guérin) in patients with non–muscle-invasive bladder cancer (NMIBC) and explore evidence for potentiation of BCG activity by MMC. Experimental Design: A 3 + 3 phase I dose-escalation trial of six weekly treatments was conducted in patients with NMIBC. MMC (10, 20, or 40 mg) was instilled intravesically for 30 minutes, followed by a 10-minute washout with gentle saline irrigation and then instillation of BCG (half or full strength) for 2 hours. Urine cytokines were monitored and compared with levels in a control cohort receiving BCG only. Murine experiments were carried out as described previously. Results: Twelve patients completed therapy, including 3 patients receiving full doses. The regimen was well tolerated with no treatment-related dose-limiting toxicities. Urinary frequency and urgency, and fatigue were common. Eleven (91.7%) patients were free of disease at a mean (range) follow-up of 21.4 (8.4–27.0) months. Median posttreatment urine concentrations of IL2, IL8, IL10, and TNFα increased over the 6-week treatment period. A greater increase in posttreatment urinary IL8 during the 6-week period was observed in patients receiving MMC + BCG compared with patients receiving BCG monotherapy. In mice, intravesical MMC + BCG skewed tumor-associated macrophages (TAM) toward a beneficial M1 phenotype. Conclusions: Instillation of sequential MMC + BCG is safe tolerable up to 40-mg MMC plus full-strength BCG. This approach could provide improved antitumor activity over BCG monotherapy by augmenting beneficial M1 TAMs. Clin Cancer Res; 21(2); 303–11. ©2014 AACR.

Acquired resistance to BRAFV600E inhibitors (BRAFi) in melanoma remains a common clinical obstacle, as is the case for any targeted drug therapy that can be developed given the plastic nature of cancers. Although there has been significant focus on the cancer cell-intrinsic properties of BRAFi resistance, the impact of BRAFi resistance on host immunity has not been explored. Here we provide preclinical evidence that resistance to BRAFi in an autochthonous mouse model of melanoma is associated with restoration of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, initially reduced by BRAFi treatment. In contrast to restoration of MDSCs, levels of T regulatory cells remained reduced in BRAFi-resistant tumors. Accordingly, tumor gene expression signatures specific for myeloid cell chemotaxis and homeostasis reappeared in BRAFi-resistant tumors. Notably, MDSC restoration relied upon MAPK pathway reactivation and downstream production of the myeloid attractant CCL2 in BRAFi-resistant melanoma cells. Strikingly, although combination checkpoint blockade (anti-CTLA-4 + anti-PD-1) was ineffective against BRAFi-resistant melanomas, the addition of MDSC depletion/blockade (anti-Gr-1 + CCR2 antagonist) prevented outgrowth of BRAFi-resistant tumors. Our results illustrate how extrinsic pathways of immunosuppression elaborated by melanoma cells dominate the tumor microenvironment and highlight the need to target extrinsic as well as intrinsic mechanisms of drug resistance. Cancer Res; 77(7); 1599–610. ©2017 AACR.

The FDA-approved mTOR inhibitor rapamycin mediates important immune effects, but its contributions to the anticancer effects of the drug are unclear. Here we report evidence that rapamycin-mediated cancer protection relies upon stimulation of γδ T cells. In a well-established mouse model of carcinogen and inflammation-driven skin carcinogenesis, IFNγ recruited γδ TCRmid T cells to the epidermis where rapamycin boosted their perforin-dependent antitumor properties. These antitumor cells were mostly Vγ5−Vγ4−Vγ1− in phenotype. IFNγ signals were required in both hematopoietic and nonhematopoietic cells for rapamycin to optimally promote epidermal infiltration of γδ TCRmid T cells, as mediated by CXCR3–CXCL10 interactions, along with the antitumor effects of these cells. In mouse xenograft models of human squamous cell carcinoma, rapamycin improved human γδ T-cell–mediated cancer cell killing. Our results identify immune mechanisms for the cancer prevention and treatment properties of rapamycin, challenging the paradigm that mTOR inhibition acts primarily by direct action on tumor cells. Cancer Res; 76(20); 5970–82. ©2016 AACR.

Purpose: To define the safety and effectiveness of T cells redirected against follicle-stimulating hormone receptor (FSHR)-expressing ovarian cancer cells. Experimental Design: FSHR expression was determined by Western blotting, immunohistochemistry, and qPCR in 77 human ovarian cancer specimens from 6 different histologic subtypes and 20 human healthy tissues. The effectiveness of human T cells targeted with full-length FSH in vivo was determined against a panel of patient-derived xenografts. Safety and effectiveness were confirmed in immunocompetent tumor-bearing mice, using constructs targeting murine FSHR and syngeneic T cells. Results: FSHR is expressed in gynecologic malignancies of different histologic types but not in nonovarian healthy tissues. Accordingly, T cells expressing full-length FSHR-redirected chimeric receptors mediate significant therapeutic effects (including tumor rejection) against a panel of patient-derived tumors in vivo. In immunocompetent mice growing syngeneic, orthotopic, and aggressive ovarian tumors, fully murine FSHR-targeted T cells also increased survival without any measurable toxicity. Notably, chimeric receptors enhanced the ability of endogenous tumor-reactive T cells to abrogate malignant progression upon adoptive transfer into naïve recipients subsequently challenged with the same tumor. Interestingly, FSHR-targeted T cells persisted as memory lymphocytes without noticeable PD-1–dependent exhaustion during end-stage disease, in the absence of tumor cell immunoediting. However, exosomes in advanced tumor ascites diverted the effector activity of this and other chimeric receptor–transduced T cells away from targeted tumor cells. Conclusions: T cells redirected against FSHR+ tumor cells with full-length FSH represent a promising therapeutic alternative against a broad range of ovarian malignancies, with negligible toxicity even in the presence of cognate targets in tumor-free ovaries. Clin Cancer Res; 23(2); 441–53. ©2016 AACR.

Purpose To investigate the safety and efficacy of durvalumab, a human monoclonal antibody that binds programmed cell death ligand-1 (PD-L1), and the role of PD-L1 expression on clinical response in patients with advanced urothelial bladder cancer (UBC). Methods A phase 1/2 multicenter, open-label study is being conducted in patients with inoperable or metastatic solid tumors. We report here the results from the UBC expansion cohort. Durvalumab (MEDI4736, 10 mg/kg every 2 weeks) was administered intravenously for up to 12 months. The primary end point was safety, and objective response rate (ORR, confirmed) was a key secondary end point. An exploratory analysis of pretreatment tumor biopsies led to defining PD-L1–positive as ≥ 25% of tumor cells or tumor-infiltrating immune cells expressing membrane PD-L1. Results A total of 61 patients (40 PD-L1–positive, 21 PD-L1–negative), 93.4% of whom received one or more prior therapies for advanced disease, were treated (median duration of follow-up, 4.3 months). The most common treatment-related adverse events (AEs) of any grade were fatigue (13.1%), diarrhea (9.8%), and decreased appetite (8.2%). Grade 3 treatment-related AEs occurred in three patients (4.9%); there were no treatment-related grade 4 or 5 AEs. One treatment-related AE (acute kidney injury) resulted in treatment discontinuation. The ORR was 31.0% (95% CI, 17.6 to 47.1) in 42 response-evaluable patients, 46.4% (95% CI, 27.5 to 66.1) in the PD-L1–positive subgroup, and 0% (95% CI, 0.0 to 23.2) in the PD-L1–negative subgroup. Responses are ongoing in 12 of 13 responding patients, with median duration of response not yet reached (range, 4.1+ to 49.3+ weeks). Conclusion Durvalumab demonstrated a manageable safety profile and evidence of meaningful clinical activity in PD-L1–positive patients with UBC, many of whom were heavily pretreated.

// Sukeshi Patel 1 , Vincent Hurez 1 , Steffan T. Nawrocki 1 , Martin Goros 1 , Joel Michalek 1 , John Sarantopoulos 1 , Tyler Curiel 1 , Devalingam Mahalingam 1 1 Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Correspondence to: Devalingam Mahalingam, email: mahalingam@uthscsa.edu Keywords: vorinostat, hydroxychloroquine, colorectal cancer, autophagy, immunity Received: May 06, 2016 Accepted: June 30, 2016 Published: July 23, 2016 ABSTRACT Hydroxychloroquine (HCQ) enhances the anti-cancer activity of the histone deacetylase inhibitor, vorinostat (VOR), in pre-clinical models and early phase clinical studies of metastatic colorectal cancer (mCRC). Mechanisms could include autophagy inhibition, accumulation of ubiquitinated proteins, and subsequent tumor cell apoptosis. There is growing evidence that autophagy inhibition could lead to improved anti-cancer immunity. To date, effects of autophagy on immunity have not been reported in cancer patients. To address this, we expanded an ongoing clinical study to include patients with advanced, refractory mCRC to evaluate further the clinical efficacy and immune effects of VOR plus HCQ. Refractory mCRC patients received VOR 400 milligrams orally with HCQ 600 milligrams orally daily, in a 3-week cycle. The primary endpoint was median progression-free survival (mPFS). Secondary endpoints include median overall survival (mOS), adverse events (AE), pharmacodynamic of inhibition of autophagy in primary tumors, immune cell analyses, and cytokine levels. Twenty patients were enrolled (19 evaluable for survival) with a mPFS of 2.8 months and mOS of 6.7 months. Treatment-related grade 3–4 AEs occurred in 8 patients (40%), with fatigue, nausea/vomiting, and anemia being the most common. Treatment significantly reduced CD4 + CD25 hi Foxp3 + regulatory and PD-1 + (exhausted) CD4 + and CD8 + T cells and decreased CD45RO-CD62L + (naive) T cells, consistent with improved anti-tumor immunity. On-study tumor biopsies showed increases in lysosomal protease cathepsin D and p62 accumulation, consistent with autophagy inhibition. Taken together, VOR plus HCQ is active, safe and well tolerated in refractory CRC patients, resulting in potentially improved anti-tumor immunity and inhibition of autophagy.

Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1–CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1–CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1–mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management. Cancer Res; 71(2); 603–13. ©2010 AACR.

CD73, originally defined as a lymphocyte differentiation antigen, is thought to function as a cosignaling molecule on T lymphocytes and an adhesion molecule that is required for lymphocyte binding to endothelium. We show here that CD73 is widely expressed on many tumor cell lines and is upregulated in cancerous tissues. Because the ecto-5′-nucleotidase activity of CD73 catalyzes AMP breakdown to immunosuppressive adenosine, we hypothesized that CD73-generated adenosine prevents tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that combining tumor CD73 knockdown and tumor-specific T-cell transfer cured all tumor-bearing mice. In striking contrast, there was no therapeutic benefit of adoptive T-cell immunotherapy in mice bearing tumors without CD73 knockdown. Moreover, blockade of the A2A adenosine receptor with a selective antagonist also augmented the efficacy of adoptive T-cell therapy. These findings identify a potential mechanism for CD73-mediated tumor immune evasion and point to a novel cancer immunotherapy strategy by targeting the enzymatic activity of tumor CD73. Cancer Res; 70(6); 2245–55

Chronic myeloid leukemia (CML) has led the way for developing rational drug development in cancer. Most cases of CML diagnosed and treated in chronic phase are extremely well controlled with imatinib monotherapy, and primary resistance is very uncommon. Even though the treatment failure rate is low, the emergence of drug resistance and the lack of eradication of the hematopoietic stem cell clone has prompted a wave of drugs to address one or both these problems. Several clinical trials (Phase I and II) of dasatinib or nilotinib in the treatment of imatinib-resistant or -intolerant Ph chromosome-positive leukemia have already reported a remarkable rate of hematologic response greater than 90% for chronic-phase patients. These drugs minimize the risk of acquired drug resistance that is particularly seen within the first 24–36 months of therapy, and can prevent early failure in these patients, Furthermore, rational, noncross-resistant combinations that include a T315I inhibitor and drugs that can eradicate the hematopoietic stem cell clone may extend the coverage to virtually all patients with bcr-abl. Here we review the 6-year impact of the ‘magic pill’, Gleevec®, (Glivec®), including the emerging problems with its treatment, the efficacy data of dasatinib and nilotinib and the very promising data of the newer generation of drugs for CML.

// Barbara Christy 1,5,6,* , Marco Demaria 7,* , Judith Campisi 7 , Jing Huang 8 , Diane Jones 1 , Sherry G. Dodds 1 , Charnae Williams 1 , Gene Hubbard 2 , Carolina B. Livi 1,9 , Xiaoli Gao 3 , Susan Weintraub 3 , Tyler Curiel 4,5 , Z. Dave Sharp 1,5,6 and Paul Hasty 1,5,6 1 Departments of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 2 Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 3 Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 4 Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 5 Cancer Therapy & Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 6 Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 7 Buck Institute for Research on Aging, Novato, CA, USA 8 Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 9 Current address: Agilent Technologies, Inc., Santa Clara, CA, USA * These authors have contributed equally to this work Correspondence to: Paul Hasty, email: // Keywords : mTOR, p53, rapamycin, longevity, SASP Received : May 18, 2015 Accepted : June 14, 2015 Published : June 23, 2015 Abstract Mechanistic target of rapamycin (mTOR) is a kinase found in a complex (mTORC1) that enables macromolecular synthesis and cell growth and is implicated in cancer etiology. The rapamycin-FK506 binding protein 12 (FKBP12) complex allosterically inhibits mTORC1. In response to stress, p53 inhibits mTORC1 through a separate pathway involving cell signaling and amino acid sensing. Thus, these different mechanisms could be additive. Here we show that p53 improved the ability of rapamycin to: 1) extend mouse life span, 2) suppress ionizing radiation (IR)-induced senescence-associated secretory phenotype (SASP) and 3) increase the levels of amino acids and citric acid in mouse embryonic stem (ES) cells. This additive effect could have implications for cancer treatment since rapamycin and p53 are anti-oncogenic.

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.

To directly dissect the role of each immune component in human tumor immunopathogenesis, we have studied the interaction between dendritic cells and T cells in the tumor environment of patients with ovarian carcinoma. We previously reported that functional plasmacytoid dendritic cells, but not functionally mature myeloid dendritic cells, accumulated in tumor microenvironments. We now show that tumor ascites macrophage-derived dendritic cells induced tumor-associated antigen–specific CD8+ T cells with effector functions. Strikingly, tumor ascites plasmacytoid dendritic cells induced interleukin-10+CCR7+CD45RO+CD8+ regulatory T cells. Four characteristics have been identified in tumor plasmacytoid dendritic cell–induced CD8+ regulatory T cells: (a) induction of CD8+ regulatory T cells is independent of CD4+CD25+ T cells; (b) CD8+ regulatory T cells significantly suppress myeloid dendritic cell–mediated tumor-associated antigen–specific T cell effector functions through interleukin-10; (c) repetitive myeloid dendritic cell stimulation can recover CD8+ regulatory T cell–mediated poor T cell proliferation, but not T cell effector function; (d) CD8+ regulatory T cells express functional CCR7, and efficiently migrate with lymphoid homing chemokine MIP-3β. Primary suppressive CCR7+CD45RO+CD8+ T cells are found in the tumor environment of patients with ovarian cancers. Thus, tumor-associated plasmacytoid dendritic cells contribute to the tumor environmental immunosuppressive network. Collectively, tumors manipulate tumor microenvironmental dendritic cell subset distribution and function to subvert tumor immunity. The data are relevant to understanding tumor immunopathology as well as reevaluating tumor immunotherapeutic strategies.

Dendritic cells (DCs) are a central element in the development of antigen-specific immune responses. The lack of a specific and efficient technique for the in vivo delivery of antigens to DCs remains a major obstacle limiting a vaccine's ability to induce an effective immune response. The efficacy of adenoviral (Ad) vectors in this regard can be enhanced through alterations in vector tropism such that DC-targeted transduction is achieved. Here, the efficiency of DC transduction by Ad vectors retargeted to DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) was studied and compared to that of Ad vectors retargeted through CD40. A comparable and significant enhancement of gene transfer to monocyte derived DCs (MDDCs) was accomplished by means of an Ad vector harboring the Fc-binding domain of Staphylococcus aureus protein A in combination with antibodies to DC-SIGN or to CD40 or with fused complexes of human Ig-Fc with their natural ligands, i.e., ICAM-3 or CD40L, respectively. Whereas CD40-targeted Ad transduction resulted in a more profound phenotypic DC maturation, DC-SIGN- and CD40-targeted Ad both induced similar levels of IL-12 secretion. These data demonstrate the usefulness of DC-SIGN as a DC-restricted targeting motif for Ad-mediated vaccination strategies.

CD4+CD25+ regulatory T cells (Tregs) mediate peripheral T-cell homeostasis and contribute to self-tolerance. Their homeostatic and pathologic trafficking is poorly understood. Under homeostatic conditions, we show a relatively high prevalence of functional Tregs in human bone marrow. Bone marrow strongly expresses functional stromal-derived factor (CXCL12), the ligand for CXCR4. Human Tregs traffic to and are retained in bone marrow through CXCR4/CXCL12 signals as shown in chimeric nonobese diabetic/severe combined immunodeficient mice. Granulocyte colony-stimulating factor (G-CSF) reduces human bone marrow CXCL12 expression in vivo, associated with mobilization of marrow Tregs to peripheral blood in human volunteers. These findings show a mechanism for homeostatic Treg trafficking and indicate that bone marrow is a significant reservoir for Tregs. These data also suggest a novel mechanism explaining reduced acute graft-versus-host disease and improvement in autoimmune diseases following G-CSF treatment.

The optimal dose of weekly docetaxel in combination with cisplatin and concomitant thoracic radiation therapy (XRT) in patients with locally advanced non-small cell lung cancer (NSCLC) is not well defined. The purpose of this study was to define the maximal tolerated dose (MTD) of docetaxel in this combination. Eligible patients had unresectable stage IIIA or IIIB NSCLC without pleural effusion. Treatment consisted of cisplatin 25 mg/m(2) plus docetaxel weekly and concomitant standard XRT for a total of 60 Gy at 200 cGy/fraction/day 5 times weekly for 6 weeks. The starting dose of docetaxel in the first cohort was 15 mg/m(2)/week. This dose was escalated by 5 mg/m(2) per cohort of 3 patients. No intrapatient dose escalation was allowed. The doses of cisplatin and XRT were not escalated. A total of 23 patients were enrolled, and 19 patients were evaluable for analysis. The first cohort (docetaxel 15 mg/m(2)/week) completed treatment without any Grade 3 or 4 toxicities. The second cohort (docetaxel 20 mg/m(2)/week) was expanded to 6 patients because of Grade 3 cough observed in 1 patient. One of 5 patients experienced Grade 3 esophagitis at the docetaxel 25 mg/m(2)/week dose level. Dose limiting toxicity consisting of Grade 3 esophagitis was reached in 4 of 5 patients receiving docetaxel at 30 mg/m(2)/week. This study determined the MTD of weekly docetaxel to be 25 mg/m(2) when combined with cisplatin 25 mg/m(2) and radiation therapy for locally advanced NSCLC. Further evaluation of this regimen in a phase II trial is underway.

Programmed death ligand (PD-L) 1 is expressed on many tumors and inhibits anti-tumor T cells through programmed death (PD)-1. Tumor PD-L1 predicts αPD-L1 treatment effects, but mechanism(s) for PD-L1- tumor response to αPD-L1 are unclear. Our studies suggest tumor-intrinsic PD-L1 signals and spatially varied PD-L1 expression may contribute to response deviation. We used PD-L1+ B16 melanoma (ctrl) and made PD-L1KO by CRISPR. αPD-L1 slowed ctrl but not PD-L1KO B16 growth in mice as expected, but PD-L1KO also responded to αPD-L1 if ctrl B16 was on the trans flank. αPD-L1 elicited similar CD3+ T cell infiltration into ctrl vs. PD-L1KO tumors, but without detectable B16-specific T cell increase. CD11b+ cell infiltration was similar in ctrl and PD-L1KO. Strikingly, NKp46+ and NK1.1+ natural killer (NK) cells infiltrated PD-L1KO > ctrl. NK cells increased significantly (~2-fold) after αPD-L1 in PD-L1KO vs. ctrl, along with NK effector functions (e.g., IFN-γ). Tumor PD-L1 altered tumor chemokines (e.g., CXCL12, CCL2) that could explain trafficking. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly > ctrl (with slightly > PD-1+ T cells) in transwells, but NK cells migrated ~2-fold more to PD-L1KO vs. ctrl (with increased activation (e.g., PD-1, CD69), also seen in vivo). We observed that tumor β2 microglobulin (B2M) expression was suppressed by tumor PD-L1. Tumor PD-L1 alteration of immune infiltrates by altering chemokines is a novel mechanism for PD-L1 TIL control. These data also indicate that tumor PD-L1 blunts NK cell infiltration in B16 and reduces B2M, which could boost NK cell anti-tumor activity. These changes correlate with rescued αPD-L1 response of PD-L1KO B16. As αPD-L1 did not increase B16-specific T cells in PD-L1low, we hypothesize that rescue of αPD-L1 response is in part NK cell-mediated. Preliminary in vivo NK cell depletion + αPD-L1 experiments implicate a role for NK cells in PD-L1KO tumor response, but more work is needed to identify mechanisms. Effects extend beyond B16 as ctrl (PD-L1+) MB49 bladder cancer cells in trans elicited αPD-L1 response of PD-L1KO MB49 tumors. Mouse breast and ovarian cancers, and human ovarian and bladder cells exhibit similar PD-L1-intrinsic effects. Finally, αPD-L1 directly altered tumor cell chemokine production in vitro, suggesting additional, novel αPD-L1 treatment mechanisms requiring more study. Our models are useful to understand αPD-L1 (and likely αPD-1) responses based on tumor PD-L1 expression and will help define strategies to improve responses in PD-L1low tumors, and possibly poorly responsive PD-L1+ tumors. Citation Format: Curtis A. Clark, Harshita B. Gupta, Alvaro Padron, Deyi Zhang, Vincent Hurez, Mary Jo Turk, Rong Li, Tyler Curiel. Tumor-intrinsic PD-L1 alters tumor chemokines, NK cell trafficking and function, and renders distant PD-L1 null tumors responsive to αPD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3696. doi:10.1158/1538-7445.AM2017-3696

Tumor initiating cells (TIC) foster treatment resistance and tumor relapse. We show here that in mouse B16 (melanoma) and ID8agg (ovarian carcinoma), and human ovarian cancer ES2, PD-L1 knockdown (shRNA) reduced TIC numbers and functions. Data shown are from 2 PD-L1lo clones/cell type. Sorted, control CD44+CD133+CD24+ (B16), CD44+CD24+ (ID8agg) and ALDHhi (ES2) TIC formed 50-75% more and bigger spheres vs. PD-L1lo TIC in vitro, consistent with reduced TIC self-renewal in PD-L1lo. In vivo, PD-L1lo B16 and ID8agg TIC exhibited reduced tumorigenicity in wild type mice, consistent with poor TIC function, but immune effects could not be excluded. B16 and ES2 PD-L1lo TIC had significantly reduced tumorigenicity versus control TIC in immune deficient NSG mice consistent with cell-autonomous, immune-independent TIC promotion by PD-L1. In further confirmation, we found reduced serial transplantability of PD-L1lo B16 TIC in NSG mice. The canonical stemness genes nanog and pou5f1 (oct4) were significantly higher in control vs. PD-L1lo TIC for B16 and ID8agg. The canonical stemness gene SOX2 was significantly higher in control vs. PD-L1lo ES2 TIC. Altogether, these data validate effects in human cells, and are consistent with PD-L1 driving TIC through stemness gene control, although specific genes differed in distinct tumors (likely due to differing mutational landscapes). mTORC1 signaling linked to stemness (assessed by raptor) was lower but mTORC2 was unaffected (rictor) in PD-L1lo vs control TIC from all 3 tumors, suggesting mTORC1 control of PD-L1-mediated TIC generation. In support, the mTORC1 inhibitor rapamycin reduced control TIC significantly (>50% vs. untreated). Strikingly, rapamycin significantly increased PD-L1lo TIC in all 3 tumors, suggesting mTORC1 effects differ by tumor PD-L1 status. Interferon (IFN)-γ similarly reduced control TIC and augmented PD-L1lo TIC numbers, suggesting the novel concept that PD-L1 alters IFN-γ signals. PD-L1 sensitization of TIC to IFN-γ and rapamycin is the first example of a molecule enhancing TIC treatment response to our knowledge. We recently reported that αPD-L1 directly reduces B16 and ID8agg proliferation in vitro and in vivo (accession no. 27671674). Preliminary data now show that αPD-L1 (and αPD-1) directly reduce TIC proliferation, and these effects differ from non-TIC. We challenge the paradigm that tumor PD-L1 is primarily an immune escape molecule and that αPD-L1 works primarily to block T cell PD-1 interactions. Citation Format: Harshita B. Gupta, Curtis A. Clark, Bin Yuan, Gangadhara Sareddy, Srilakshmi Pandeswara, Alvaro S. Padron, Vincent Hurez, Jose-Conejo Garcia, Ratna Vadlamudi, Rong Li, Tyler J. Curiel. Cell-intrinsic PD-L1 directs tumor initiating cell fate and rapamycin and interferon-γ response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5649. doi:10.1158/1538-7445.AM2017-5649