Quantitative systems pharmacology model-based investigation of adverse gastrointestinal events associated with prolonged treatment with PI3-kinase inhibitors.

Several PI3K inhibitors are in clinical development for the treatment of various forms of cancers, including pan-PI3K inhibitors targeting all four PI3K isoforms (α, ß, γ, and δ), and isoform-selective inhibitors. Diarrhea and immune-mediated colitis are among the adverse events observed with PI3K inhibition which limits the maximal tolerated dose. A quantitative systems pharmacology model was developed to investigate PI3K-inhibitor-induced colitis. The effects of individual PI3K isoforms on relevant cellular pathways were incorporated into a mechanistic representation of mucosal inflammation. A virtual clinical population captures the observed clinical variability in the onset timing and rates of diarrhea and colitis for seven clinically tested PI3K inhibitors. Model-based analysis suggests that colitis development is governed by both the inhibition of PI3Kδ, which drives T cell differentiation and proliferation, and PI3Kα, which regulates epithelial barrier integrity. Specifically, when PI3Kα is inhibited below a given threshold, epithelial barrier dysfunction precipitates an exaggerated T effector response due to PI3Kδ-inhibition, leading to risk of diarrhea and colitis. This synergy explains why the lowest diarrhea and colitis rates are seen with the weakest PI3Kδ inhibition (alpelisib), and higher rates are seen with strong PI3Kδ inhibition if PI3Kα is even mildly inhibited (e.g., idelalisib), whereas strong PI3Kδ inhibition in the absence of PI3Kα inhibition does not result in high colitis rates (umbralisib). Thus, the model-based analysis suggests that PI3Kα and δ inhibition play unique but synergistic roles in driving colitis. Finally, we explore if and how dose-regimen might influence colitis rates for molecules that inhibit both PI3Kα and PI3Kδ.

IFNα Augments Clinical Efficacy of Regulatory T-cell Depletion with Denileukin Diftitox in Ovarian Cancer.

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.

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

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

Percutaneous BCG enhances innate effector antitumor cytotoxicity during treatment of bladder cancer: a translational clinical trial.

Background: Intravesical bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for treating patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, relapse rates remain high and BCG unresponsive NMIBC often requires bladder removal. Preclinical data suggest that priming with percutaneous BCG vaccine could improve response to intravesical BCG. Methods: A single-arm trial (NCT02326168) was performed to study the safety, immunogenicity, and preliminary efficacy of priming. Percutaneous BCG was given 21 days prior to intravesical BCG instillation in patients (n = 13) with high-risk NMIBC. Immune responses were monitored and compared to a sequentially enrolled cohort of nine control patients receiving only intravesical BCG. The effect of BCG on natural killer (NK) and γδ T cell in vitro cytotoxicity was tested. γδ T cell subsets were determined by T cell receptor gene expression with NanoString. Results: Priming was well tolerated and caused no grade ≥3 adverse events. The 3-month disease-free rate for prime patients was 85% (target goal ≥ 75%). Priming boosted BCG-specific immunity at 3 months and increased the activation status of in vitro expanded circulating NK and γδ T cells and their cytotoxicity against bladder cancer cells through receptor NKG2D. BCG enhanced the cytotoxicity of NK and γδ T cells against K562, RT4, and UM-UC6 but not against T24, UM-UC-3, or UM-UC-14 cells. Infiltrating γδ T cell subsets identified in the bladder includes γ9δ2 and γ8δ2. Conclusions: BCG priming is safe and tolerable. Poor sensitivity to NK and γδ T cell cytotoxicity by some bladder tumors represents a potential BCG-resistance mechanism.

Rapamycin Prevents Surgery-Induced Immune Dysfunction in Patients with Bladder Cancer.

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients (P = 0.008). Rapamycin reduced surgery-induced T-cell exhaustion in patients, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1)-expressing T cells. Grade 3 to 4 adverse event rates were similar between groups, but rapamycin-treated patients had a higher rate of wound complications versus controls. In conclusion, surgery promoted bladder cancer metastasis and decreased the efficacy of postoperative bladder cancer immunotherapy. Low-dose (3 mg daily) oral rapamycin has favorable pharmacodynamic and immune modulating activity in surgical patients and has the potential to decrease surgery-induced immune dysfunction.

Intratumoral CD56bright natural killer cells are associated with improved survival in bladder cancer.

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 CD56bright and CD56dim 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 CD56bright NK cells showed increased cytokine production and cytotoxicity compared to their CD56dim counterparts and were associated with improved CSS and OS independent of pathologic tumor stage. On the other hand, CD56dim 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 CD56bright NK cells are functional and prognostically relevant whereas CD56dim NK cells are dysfunctional and prevalent in higher stage tumors. Thus, CD56bright NK cells are promising targets in BC.

Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model.

Cancer immunotherapy has shown remarkable recent progress. Immune checkpoint blocking antibodies have become the most successful anti-cancer agent class ever developed, with six distinct agents approved since 2011 for a wide variety of cancers. Although age is the biggest risk factor for cancer (aside from selected early-onset pediatric cancers), these agents were tested pre-clinically in young hosts, and there is remarkably little published on the effects of host age on treatment outcomes in pre-clinical studies or human clinical trials. The three principal immune checkpoints against which blocking antibodies have been FDA-approved for human use are CTLA-4, PD-1 and PD-L1. We used a mouse model of transplantable, orthotopic B16 melanoma to test age effects of treatments with anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibodies. All three agents were highly effective in treating young tumor-bearing hosts as expected. Anti-PD-L1 as a single agent had no effect on tumor growth in aged hosts, anti-CTLA-4 had detectable, modest effects and anti-PD-1 was essentially as effective in aged as in young hosts, the first single agent we have identified not to lose efficacy with age in this model. Other important differences in young versus aged hosts included lack of anti-CTLA-4-mediated depletion of intratumor regulatory T cells in aged hosts and poorer ability of all three agents to activate T cells in aged versus young hosts. Anti-CTLA-4 efficacy appeared to improve when combined with anti-PD-L1. Regulatory T cell depletion with FDA-approved denileukin diftitox did not improve treatment by any single agent. Aged mice tolerated treatments as well as young mice without obvious toxicities at equivalent doses.

Considerations for successful cancer immunotherapy in aged hosts.

Improvements in understanding cancer immunopathogenesis have now led to unprecedented successes in immunotherapy to treat numerous cancers. Although aging is the most important risk factor for cancer, most pre-clinical cancer immunotherapy studies are undertaken in young hosts. This review covers age-related immune changes as they affect cancer immune surveillance, immunopathogenesis and immune therapy responses. Declining T cell function with age can impede efficacy of age-related cancer immunotherapies, but examples of successful approaches to breach this barrier have been reported. It is further recognized now that immune functions with age do not simply decline, but that they change in potentially detrimental ways. For example, detrimental immune cell populations can become predominant during aging (notably pro-inflammatory cells), the prevalence or function of suppressive cells can increase (notably myeloid derived suppressor cells), drugs can have age-specific effects on immune cells, and attributes of the aged microenvironment can impede or subvert immunity. Key advances in these and related areas will be reviewed as they pertain to cancer immunotherapy in the aged, and areas requiring additional study and some speculations on future research directions will be addressed. We prefer the term Age Related Immune Dysfunction (ARID) as most encompassing the totality of age-associated immune changes.

Biphasic Rapamycin Effects in Lymphoma and Carcinoma Treatment.

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.

Tumor-Intrinsic PD-L1 Signals Regulate Cell Growth, Pathogenesis, and Autophagy in Ovarian Cancer and Melanoma.

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.

Immune-Stimulatory Effects of Rapamycin Are Mediated by Stimulation of Antitumor γδ T Cells.

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.

Vorinostat and hydroxychloroquine improve immunity and inhibit autophagy in metastatic colorectal cancer.

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+CD25hiFoxp3+ 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.

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

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

Chronic mTOR inhibition in mice with rapamycin alters T, B, myeloid, and innate lymphoid cells and gut flora and prolongs life of immune-deficient mice.

The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age-related debilities including increasing antigen-specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long-term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T- and B-lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer- and infection-prone mice supporting disease mitigation as a mechanism for mTOR suppression-mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension.

Prevention of carcinogen and inflammation-induced dermal cancer by oral rapamycin includes reducing genetic damage.

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, IL1ß, IL6, or TNFα. In support of lack of mTORC1 inhibition, eRapa did not reduce numbers or proliferation of CD45(-)CD34(+)CD49f(mid) 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.

Immunotherapy for ovarian cancer.

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

Sequential intravesical mitomycin plus Bacillus Calmette-Guérin for non-muscle-invasive urothelial bladder carcinoma: translational and phase I clinical trial.

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.

eRapa restores a normal life span in a FAP mouse model.

Mutation of a single copy of the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer. Apc(Min/+) mice exhibit multiple intestinal neoplasia (MIN) that causes anemia and death from bleeding by 6 months. Mechanistic target of rapamycin complex 1 (mTORC1) inhibitors were shown to improve Apc(Min/+) mouse survival when administered by oral gavage or added directly to the chow, but these mice still died from neoplasia well short of a natural life span. The National Institute of Aging Intervention Testing Program showed that enterically targeted rapamycin (eRapa) extended life span for wild-type genetically heterogeneous mice in part by inhibiting age-associated cancer. We hypothesized that eRapa would be effective in preventing neoplasia and extend survival of Apc(Min/+) mice. We show that eRapa improved survival of Apc(Min/+) mice in a dose-dependent manner. Remarkably, and in contrast to previous reports, most of the Apc(Min/+) mice fed 42 parts per million eRapa lived beyond the median life span reported for wild-type syngeneic mice. Furthermore, chronic eRapa did not cause detrimental immune effects in mouse models of cancer, infection, or autoimmunity, thus assuaging concerns that chronic rapamycin treatment suppresses immunity. Our studies suggest that a novel formulation (enteric targeting) of a well-known and widely used drug (rapamycin) can dramatically improve its efficacy in targeted settings. eRapa or other mTORC1 inhibitors could serve as effective cancer preventatives for people with FAP without suppressing the immune system, thus reducing the dependency on surgery as standard therapy.

TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production.

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

Mitigating age-related immune dysfunction heightens the efficacy of tumor immunotherapy in aged mice.

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.