IDH1 inhibition potentiates chemotherapy efficacy in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a five-year overall survival rate of just 13%, and development of chemotherapy resistance is nearly universal. PDAC cells overexpress wild-type IDH1 that can enable them to overcome metabolic stress, suggesting it could represent a therapeutic target in PDAC. Here, we found that anti-IDH1 therapy enhanced the efficacy of conventional chemotherapeutics. Chemotherapy treatment induced ROS and increased TCA cycle activity in PDAC cells, along with the induction of wild-type IDH1 expression as a key resistance factor. IDH1 facilitated PDAC survival following chemotherapy treatment by supporting mitochondrial function and antioxidant defense to neutralize reactive oxygen species through the generation of alpha-ketoglutarate and NADPH, respectively. Pharmacologic inhibition of wild-type IDH1 with ivosidenib synergized with conventional chemotherapeutics in vitro and potentiated the efficacy of sub-therapeutic doses of these drugs in vivo in murine PDAC models. This promising treatment approach is translatable through available and safe oral inhibitors and provides the basis of an open and accruing clinical trial testing this combination (NCT05209074).

Increased glucose availability sensitizes pancreatic cancer to chemotherapy.

Pancreatic Ductal Adenocarcinoma (PDAC) is highly resistant to chemotherapy. Effective alternative therapies have yet to emerge, as chemotherapy remains the best available systemic treatment. However, the discovery of safe and available adjuncts to enhance chemotherapeutic efficacy can still improve survival outcomes. We show that a hyperglycemic state substantially enhances the efficacy of conventional single- and multi-agent chemotherapy regimens against PDAC. Molecular analyses of tumors exposed to high glucose levels reveal that the expression of GCLC (glutamate-cysteine ligase catalytic subunit), a key component of glutathione biosynthesis, is diminished, which in turn augments oxidative anti-tumor damage by chemotherapy. Inhibition of GCLC phenocopies the suppressive effect of forced hyperglycemia in mouse models of PDAC, while rescuing this pathway mitigates anti-tumor effects observed with chemotherapy and high glucose.

Targeting wild-type IDH1 enhances chemosensitivity in pancreatic cancer.

Pancreatic cancer (PC) is one of the most aggressive types of cancer, with a five-year overall survival rate of 11% among all-comers. Current systemic therapeutic options are limited to cytotoxic chemotherapies which have limited clinical efficacy and are often associated with development of drug resistance. Analysis of The Cancer Genome Atlas showed that wild-type isocitrate dehydrogenase (wtIDH1) is overexpressed in pancreatic tumors. In this study, we focus on the potential roles of wtIDH1 in pancreatic cancer chemoresistance. We found that treatment of pancreatic cancer cells with chemotherapy induced expression of wtIDH1, and this serves as a key resistance factor. The enzyme is protective to cancer cells under chemotherapy-induced oxidative stress by producing NADPH and alpha-ketoglutarate to maintain redox balance and mitochondrial function. An FDA-approved mutant IDH1 inhibitor, ivosidenib (AG-120), is actually a potent wtDH1 inhibitor under a nutrient-deprived microenvironment, reflective of the pancreatic cancer microenvironment. Suppression of wtIDH1 impairs redox balance, results in increased ROS levels, and enhances chemotherapy induced apoptosis in pancreatic cancer vis ROS damage in vitro. In vivo experiments further revealed that inhibiting wtIDH1 enhances chemotherapy anti-tumor effects in patient-derived xenografts and murine models of pancreatic cancer. Pharmacologic wtIDH1 inhibition with ivosidenib represents an attractive option for combination therapies with cytotoxic chemotherapy for patients with pancreatic cancer. Based on these data, we have initiated phase Ib trial combining ivosidenib and multi-agent chemotherapy in patients with pancreatic cancer (NCT05209074).

IDO1 Is a Therapeutic Target for Pancreatic Cancer-Associated Depression.

Metabolites of tryptophan degradation are known to alter mood. Their effects have only been superficially examined in the context of pancreatic cancer. Herein, we study the role of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme important in the conversion of tryptophan to kynurenine, in a murine model of pancreatic cancer-associated depression. Behavioral tests (open field, forced swim, tail suspension, and elevated plus maze) and biochemical assays (LC-MS metabolomics) were used to characterize a depressive-phenotype in tumor-bearing mice (relative to non-tumor-bearing mice). In addition, we determine whether pharmacologic blockade of IDO1 affects mood in tumor-bearing mice. Immunocompetent mice bearing orthotopic pancreatic tumors exhibit depressive-like behavior relative to non-tumor-bearing mice. Pancreatic tumors strongly express IDO1. Consequently, serum kynurenine levels in tumor-bearing mice are elevated relative to non-tumor-bearing mice. Tumor-bearing mice treated with epacadostat, an IDO1 inhibitor, exhibited improved mood relative to mice receiving vehicle. There was a 95% reduction in serum kynurenine levels in mice receiving epacadostat relative to mice treated with vehicle. As confirmatory evidence of on-target activity, tumors of mice treated with epacadostat exhibited a compensatory increase in IDO1 protein levels. Escitalopram, an approved antidepressant, was ineffective at improving mood in tumor-bearing mice as measured by behavioral assays and did not affect kynurenine levels. Neither epacadostat, nor escitalopram, affected overall survival relative to vehicle. Mice with pancreatic cancer exhibit depressive-like behavior. Epacadostat was effective as an antidepressant for pancreatic cancer-associated depression in mice. These data offer a rationale to consider IDO1 inhibition as a therapeutic strategy to mitigate depressive symptoms in patients with pancreatic cancer.

Wild-type IDH1 inhibition enhances chemotherapy response in melanoma.

BACKGROUND: Alternative treatment strategies in melanoma beyond immunotherapy and mutation-targeted therapy are urgently needed. Wild-type isocitrate dehydrogenase 1 (wtIDH1) has recently been implicated as a metabolic dependency in cancer. The enzyme protects cancer cells under metabolic stress, including nutrient limited conditions in the tumor microenvironment. Specifically, IDH1 generates NADPH to maintain redox homeostasis and produces α-ketoglutarate to support mitochondrial function through anaplerosis. Herein, the role of wtIDH1 in melanoma is further explored. METHODS: The expression of wtIDH1 was determined by qRT-PCR, and Western blot in melanoma cell lines and the effect of wtIDH1 on metabolic reprogramming in melanoma was interrogated by LC-MS. The impact of wtIDH1 inhibition alone and in combination with chemotherapy was determined in cell culture and mouse melanoma models. RESULTS: Melanoma patients express higher levels of the wtIDH1 enzyme compared to normal skin tissue, and elevated wtIDH1 expression portends poor patient survival. Knockdown of IDH1 by RNA interference inhibited cell proliferation and migration under low nutrient levels. Suppression of IDH1 expression in melanoma also decreased NADPH and glutathione levels, resulting in increased reactive oxygen species. An FDA-approved inhibitor of mutant IDH1, ivosidenib (AG-120), exhibited potent anti-wtIDH1 properties under low magnesium and nutrient levels, reflective of the tumor microenvironment in natura. Thus, similar findings were replicated in murine models of melanoma. In light of the impact of wtIDH1 inhibition on oxidative stress, enzyme blockade was synergistic with conventional anti-melanoma chemotherapy in pre-clinical models. CONCLUSIONS: These results demonstrate the clinical potential of wtIDH1 inhibition as a novel and readily available combination treatment strategy for patients with advanced and refractory melanoma. Schematic shows increased wild-type IDH1 expression and activity as an adaptive response to metabolic stress induced by chemotherapy.

Limited nutrient availability in the tumor microenvironment renders pancreatic tumors sensitive to allosteric IDH1 inhibitors.

Nutrient-deprived conditions in the tumor microenvironment (TME) restrain cancer cell viability due to increased free radicals and reduced energy production. In pancreatic cancer cells a cytosolic metabolic enzyme, wild-type isocitrate dehydrogenase 1 (wtIDH1), enables adaptation to these conditions. Under nutrient starvation, wtIDH1 oxidizes isocitrate to generate α-ketoglutarate (αKG) for anaplerosis and NADPH to support antioxidant defense. In this study, we show that allosteric inhibitors of mutant IDH1 (mIDH1) are potent wtIDH1 inhibitors under conditions present in the TME. We demonstrate that low magnesium levels facilitate allosteric inhibition of wtIDH1, which is lethal to cancer cells when nutrients are limited. Furthermore, the Food & Drug Administration (FDA)-approved mIDH1 inhibitor ivosidenib (AG-120) dramatically inhibited tumor growth in preclinical models of pancreatic cancer, highlighting this approach as a potential therapeutic strategy against wild-type IDH1 cancers.

Prodromal depression and anxiety are associated with worse treatment compliance and survival among patients with pancreatic cancer.

OBJECTIVE: To determine the frequency of depression or anxiety preceding a diagnosis of pancreatic cancer (PC). Further, to examine the association of PC-associated depression or anxiety with treatment compliance and survival. METHODS: 856 patients with PC from a single institution were identified using International Classification of Diseases (ICD) codes. For each case, two non-cancer age- and sex-matched controls were included. Dates of depression or anxiety diagnosis identified using ICD codes were compared to the date of PC diagnosis. The medical record was queried to further explore psychiatric symptoms. Multivariable analyses were performed to examine if prediagnosis depression or anxiety was associated with receipt of treatment or survival. RESULTS: A greater proportion of patients with PC experienced depression or anxiety in the year preceding diagnosis than the overall frequency in controls (4.6% vs. 2.6%, p = 0.005) based on ICD codes. Patients with PC exhibited signs of prodromal depression or anxiety based on ICD codes, clinical documentation of psychiatric symptoms, or initiation of new psychiatric medications more often than controls (20.7% vs. 6.7%, p < 0.001). Prediagnosis depression or anxiety was associated with a reduced likelihood of receiving chemotherapy (OR = 0.58, p = 0.04). There was an associated decrease in overall survival among patients with metastatic disease who experienced depression or anxiety before PC diagnosis (HR = 1.32, p = 0.04). CONCLUSIONS: The frequency of depression or anxiety among patients with PC was higher than the general population. Prediagnosis psychiatric symptoms were associated with reduced chemotherapy utilization and worse overall survival. Thus, timely identification and treatment of these symptoms may improve outcomes.

Clinical development of IDH1 inhibitors for cancer therapy.

Isocitrate dehydrogenase 1 (IDH1) has been investigated as a promising therapeutic target in select cancers with a mutated version of the enzyme (mtIDH1). With only one phase III trial published to date and two indications approved for routine clinical use by the FDA, we reviewed the entire clinical trial portfolio to broadly understand mtIDH1 inhibitor activity in patients. We queried PubMed.gov and ClinicalTrials.gov to identify published and ongoing clinical trials related to IDH1 and cancer. Progression-free survival (PFS), overall survival (OS), 2-hydroxyglutarate levels, and adverse events were summarized. To date, ten clinical trials investigating mtIDH1 inhibitors among patients with diverse malignancies (cholangiocarcinoma, acute myeloid leukemia, chondrosarcoma, glioma) have been published. Almost every trial (80%) has investigated ivosidenib. In multiple phase I trials, ivosidenib treatment resulted in promising radiographic and biochemical responses with improved survival outcomes (relative to historic data) among patients with both solid and hematologic mtIDH1 malignancies. Among patients enrolled in a phase III trial with advanced cholangiocarcinoma, ivosidenib resulted in a PFS rate of 32% at 6 months, as compared to 0% with placebo. There was a 5.2 month increase in OS with ivosidenib relative to placebo, after considering crossover. The treatment-specific grade ≥3 adverse event rate of ivosidenib was 2%-26% among all patients, and was just 3.6% among 284 patients who had a solid tumor across four trials. Although <1% of malignancies harbor IDH1 mutations, small molecule mtIDH1 inhibitors, namely ivosidenib, appear to be biologically active and well tolerated in patients with solid and hematologic mtIDH1 malignancies.

Systemic administration of ß-glucan of 200 kDa modulates melanoma microenvironment and suppresses metastatic cancer.

Converting an immunosuppressive melanoma microenvironment into one that favors the induction of antitumor immunity is indispensable for effective cancer immunotherapy. In the current study we demonstrate that oat-derived ß-(1-3)-(1-4)-glucan of 200 kDa molecular size (BG34-200) previously shown to mediate direct interaction with macrophages could alter the immune signature within melanoma microenvironment. Systemic administration of BG34-200 resulted in reversion of tolerant melanoma microenvironment to an immunogenic one that allows M1-type activation of macrophages, the induction of pro-inflammatory cytokines/chemokines including IFN-γ, TNF-α, CXCL9, and CXCL10, and enhanced IRF1 and PD-L1 expression. In turn, BG34-200 induced a superior antitumor response against primary and lung metastatic B16F10 melanoma compared to untreated controls. The enhanced tumor destruction was accompanied with significantly increased tumor infiltration of CD4+ and CD8+ T cells as well as elevated IFN-γ in the tumor sites. Systemic administration of BG34-200 also provoked systemic activation of tumor draining lymph node T cells that recognize antigens naturally expressing in melanoma (gp100/PMEL). Mechanistic studies using CD11b-knockout (KO), CD11 c-DTR transgenic mice and nude mice revealed that macrophages, DCs, T cells and NK cells were all required for the BG34-200-induced therapeutic benefit. Studies using IFN-γ-KO transgenic mice showed that IFN-γ was essential for the BG34-200-elicited antitumor response. Beyond melanoma, the therapeutic efficacy of BG34-200 and its immune stimulating activity were demonstrated in a mouse model of osteosarcoma. Together, BG34-200 is highly effective in modulating antitumor immunity. Our data support the potential therapeutic use of this novel immune modulator in the treatment of metastatic melanoma.

Identification of Melanoma-reactive CD4+ T-Cell Subsets From Human Melanoma Draining Lymph Nodes.

Our laboratory has previously demonstrated that melanoma draining lymph node (MDLN) samples from stage III patients contained both CD4 and CD8 T cells that can be readily expanded to mediate tumor cell apoptosis in vitro and improve survival in mice bearing human melanoma xenografts. In this study, we investigated whether MDLN T cells contain melanoma-reactive CD4 T-cell compartment and what they are. To test this, we performed multiparametric (11-color and 6-color) fluorescence-activated cell sorting analyses to monitor phenotypic and functional property of CD4 T cells in response to melanoma cell antigen reexposure. Our results have demonstrated that the antigen reexposure could result in a generation of CD4CCR7CD62LCD27 T-cell subsets with various effector cell-like properties. Within the CD4CCR7CD62LCD27 T-cell compartment, in response to antigen reexposure, some of the cells expressed significantly upregulated CD40L and/or CXCR5, and some of them expressed significantly upregulated interleukin-2 and/or tumor necrosis factor-α. This may suggest the existence of melanoma-reactive CD4 "effector-precursor" cells within the expanded MDLN cells and their differentiation into various effector lineages in response to antigen restimulation. Recent clinical trials have demonstrated that effective adoptive cellular immunotherapy maybe enhanced by antigen-specific CD4 T cells. Therefore, results of this study may significantly benefit innovative design of +adoptive cellular immunotherapy that can potentially mediate enhanced and durable clinical responses.

Requirement of Innate Immunity in Tumor-Bearing Mice Cured by Adoptive Immunotherapy Using Tumor-Draining Lymph Nodes.

BACKGROUND: The purpose of this study was to determine the cellular effectors of both the adoptively transferred cells and the tumor-bearing host that participate in the antitumor response to adoptive immunotherapy using culture-activated tumor-draining lymph nodes (TDLNs). METHODS: TDLNs harvested from mice with 4T1 carcinoma cells were fractionated to derive the L-selectin(low) subpopulation and activated ex vivo prior to in vitro cytokine release assays and adoptive transfer into BALB/c mice bearing 3-day established subcutaneous tumors. Tumor-bearing recipients were SCID (lacking T, B, and NK cells), Rag2 deficient (lacking T and B cells), and wild-type BALB/c mice. RESULTS: Culture-activated L-selectin(low) 4T1 TDLN from BALB/c mice secreted significant levels of interferon-gamma in response to 4T1 but not control tumor cells in vitro. CD4 cells within the adoptively transferred effector cell population contributed significantly to the antitumor effect in vivo. Culture-activated L-selectin(low) TDLNs from BALB/c wild-type mice were able to cure Rag2 deficient but not SCID mice bearing 4T1 subcutaneous tumors, suggesting a requirement of NK cells within the innate immune system of the tumor-bearing host during the antitumor response. CONCLUSIONS: These results identify the cellular effectors involved in tumor regression following adoptive transfer and demonstrate the requirement for intact innate immunity within the tumor-bearing host.

T Cells Derived From Human Melanoma Draining Lymph Nodes Mediate Melanoma-specific Antitumor Responses In Vitro and In Vivo in Human Melanoma Xenograft Model.

It has been established in murine models that lymph nodes draining a progressively growing tumor contain antigen-specific T cells capable of mediating protective immune responses upon adoptive transfer. However, naturally occurring human tumor-draining lymph nodes (TDLNs) have yet to be fully investigated. In this study, we analyzed TDLNs from patients with stage III melanoma who were undergoing routine lymph node dissection. Following short-term (14 d) culture activation with anti-CD3/anti-CD28 microbeads and expansion in low concentrations of IL-2, the melanoma-draining lymph node (MDLN) cells were ∼ 60% CD4-activated and ∼ 40% CD8-activated T cells. The activated MDLN cells demonstrated reactivity in response to overlapping peptides spanning the sequence of 4 different known melanoma antigens MAGEA1, Melan-A/MART-1, NY-ESO-1, and Prame/OIP4, suggesting the presence of melanoma-specific T cells. Coculture of activated MDLN T cells with cancer cells in vitro resulted in preferential apoptosis of human cancer cell lines that were cocultured with T cells with high degree of MHC matching. Adoptive transfer of MDLN T cells with high degree of MHC matching to A375 to mice-bearing human A375 melanoma xenografts resulted in dose-dependent improvement in survival. Although prior human studies have demonstrated the immune responses within melanoma vaccine-draining lymph nodes, this study presents evidence for the first time that naturally occurring human MDLN samples contain melanoma-experienced CD4 and CD8 T cells that can be readily cultured and expanded to mediate protective immune responses both in vitro and in vivo in a human melanoma xenograft model.

Expansion of melanoma-specific T cells from lymph nodes of patients in stage III: implications for adoptive immunotherapy in treating cancer.

BACKGROUND: Adoptive immunotherapy for patients with metastatic melanoma has yielded encouraging results. However, methods of expanding melanoma-specific T cells from stage III are limited. The objective of this study was to determine whether melanoma-specific T cells could be generated from the melanoma-draining lymph nodes (MDLNs) of patients in stage III. METHODS: Patients in stage III who were undergoing completion lymphadenectomy were enrolled into a protocol approved by the institutional review board. MDLN cells were tested for ability to undergo cryopreservation, expand ex vivo in IL-2 or IL-2 and IL-7, and mediate melanoma-specific antitumor responses in vitro. RESULTS: Cryopreservation produced no significant differences from fresh cultures in terms of cell growth and cellular phenotype. IL-2 and IL-2/IL-7 cultures resulted in similar growth rates, and functional studies revealed the presence of T cells that secreted interferon gamma in response to melanoma antigen peptides. Both IL-2- and IL-2/IL-7-cultured MDLN cells mediated significant apoptosis of human melanoma cell lines as compared to breast and brain tumor lines in vitro. Overall, there did not seem to be a benefit of adding IL-7. Both CD4+ and CD8+ T cells appear to mediate tumor cell apoptosis. CONCLUSION: This study demonstrates that melanoma antigen-specific T cells can be generated from regional melanoma-draining lymph nodes and expanded ex vivo from patients with stage III disease.

Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells.

T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4+ and CD8+ T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8+ subset, or IL-7 + IL-23 for the CD4+ subset. A broad repertoire of TCR Vbeta families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8+ T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4+ T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8+ T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4+ and CD8+ T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.

CDR3 spectratyping identifies clonal expansion within T-cell subpopulations that demonstrate therapeutic antitumor activity.

BACKGROUND: Antigen-specific T cells undergoing clonal expansion share common rearrangements of the variable complementary determining region 3 (CDR3) of the T-cell receptor (TCR), which can be identified using polymerase chain reaction-based V beta (VB) spectratyping. The purpose of this study was to determine whether CDR3 spectratyping identifies clonal expansion within tumor-draining lymph node (TDLN) subpopulations with antitumor therapeutic activity. METHODS: Recently sensitized T cells from 4T1 murine mammary carcinoma TDLN were fractionated based on CD62L (L-selectin) surface expression before RNA isolation and culture. L-selectinlow and L-selectinhigh TDLN were analyzed for T-cell receptor usage by immunophenotyping and CDR3 spectratyping, and then culture activated with anti-CD3/IL-2 to assess therapeutic efficacy after adoptive transfer. RESULTS: Adoptive transfer experiments confirmed that mice treated with culture-activated L-selectinlow TDLN cells exhibited delayed subcutaneous tumor growth and prolonged survival as compared to control or L-selectinhigh-treated mice (P < .01). CDR3 spectratyping demonstrated oligoclonal skewing of the CDR3 regions within several VB families including VB3, VB5.2, and VB17 in L-selectinlow but not in L-selectinhigh TDLN. Although fluorescence-activated cell sorter analysis demonstrated the highest percentage of cells expressing VB13 usage in both populations, CDR3 spectratyping did not identify the presence of clonal expansion. CONCLUSIONS: These data suggest that CDR3 spectratyping may be useful in identifying T cells undergoing clonal expansion that demonstrate antitumor therapeutic activity.