Inhibition Of Pyruvate Dehydrogenase Research Articles

The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells.

In recent years, growing evidence has indicated the pivotal role of mitochondria in mast cell immunological activation. We have previously reported a decrease in degranulation and cytokine secretion following the inhibition of pyruvate dehydrogenase (PDH) either by CPI-613 (PDH inhibitor/anti-cancer drug) or through its interaction with mitochondrial microphthalmia-associated transcription factor (MITF). In the present study, we further explored the role played by mitochondrial MITF in mast cell exocytosis using rat basophil leukemia cells [RBL], as well as mouse bone marrow-derived mast cells (BMMCs). Here, we report that mast cell degranulation, cytokine secretion and oxidative phosphorylation (OXPHOS) activities were associated with phosphorylation of Serine 73 of mitochondrial MITF, controlled by extracellular signals regulated by protein kinase (ERK1/2) activity. Also, we report here that decreased OXPHOS activity following ERK1/2 inhibition (U0126 treatment) during IgE-Ag activation was mediated by the dephosphorylation of Serine 73 mitochondrial MITF, which inhibited its association with PDH. This led to a reduction in mast cell reactivity. In addition, a phosphorylation-mimicking mitochondrial MITF-S73D positively regulated the mitochondrial activity, thereby supporting mast cell degranulation. Thus, the present research findings highlight the prominence of mitochondrial MITF Serine 73 phosphorylation in immunologically activated mast cells.

In Vitro and In Vivo Metabolism of a Novel Antimitochondrial Cancer Metabolism Agent, CPI-613, in Rat and Human.

CPI-613, an inhibitor of pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) enzymes, is currently in development for the treatment of pancreatic cancer, acute myeloid leukemia, and other cancers. CPI-613 is an analog of lipoic acid, an essential cofactor for both PDH and KGDH. Metabolism and mass balance studies were conducted in rats after intravenous administration of [14C]-CPI-613. CPI-613 was eliminated via oxidative metabolism followed by excretion of the metabolites in feces (59%) and urine (22%). β-Oxidation was the major pathway of elimination for CPI-613. The most abundant circulating components in rat plasma were those derived from β-oxidation. In human hepatocytes, CPI-613 mainly underwent β-oxidation (M1), sulfur oxidation (M2), and glucuronidation (M3). The Michaelis-Menten kinetics (Vmax and Km) of the metabolism of CPI-613 to these three metabolites predicted the fraction metabolized leading to the formation of M1, M2, and M3 to be 38%, 6%, and 56%, respectively. In humans, after intravenous administration of CPI-613, major circulating species in plasma were the parent and the β-oxidation derived products. Thus, CPI-613 metabolites profiles in rat and human plasma were qualitatively similar. β-Oxidation characteristics and excretion patterns of CPI-613 are discussed in comparison with those reported for its endogenous counterpart, lipoic acid. SIGNIFICANCE STATEMENT: This work highlights the clearance mechanism of CPI-613 via β-oxidation, species differences in their ability to carry out β-oxidation, and subsequent elimination routes. Structural limitations for completion of terminal cycle of β-oxidation is discussed against the backdrop of its endogenous counterpart lipoic acid.

Dichloroacetate reactivates pyruvate-supported peroxide removal by liver mitochondria and prevents NAFLD aggravation in NAD(P)+ transhydrogenase-null mice consuming a high-fat diet

The mechanisms by which a high-fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunction and redox imbalance. The functional loss of the enzyme NAD(P)+ transhydrogenase, a main source of mitochondrial NADPH, results in impaired mitochondrial peroxide removal, pyruvate dehydrogenase inhibition by phosphorylation, and progression of NAFLD in HFD-fed mice. The present study aimed to investigate whether pharmacological reactivation of pyruvate dehydrogenase by dichloroacetate attenuates the mitochondrial redox dysfunction and the development of NAFLD in NAD(P)+ transhydrogenase-null (Nnt−/−) mice fed an HFD (60% of total calories from fat). For this purpose, Nnt−/− mice and their congenic controls (Nnt+/+) were fed chow or an HFD for 20 weeks and received sodium dichloroacetate or NaCl in the final 12 weeks via drinking water. The results showed that HFD reduced the ability of isolated liver mitochondria from Nnt−/− mice to remove peroxide, which was prevented by the dichloroacetate treatment. HFD-fed mice of both Nnt genotypes exhibited increased body and liver mass, as well as a higher content of hepatic triglycerides, but dichloroacetate treatment attenuated these abnormalities only in Nnt−/− mice. Notably, dichloroacetate treatment decreased liver pyruvate dehydrogenase phosphorylation levels and prevented the aggravation of NAFLD in HFD-fed Nnt−/− mice. Conversely, dichloroacetate treatment elicited moderate hepatocyte ballooning in chow-fed mice, suggesting potentially toxic effects. We conclude that the protection against HFD-induced NAFLD by dichloroacetate is associated with its role in reactivating pyruvate dehydrogenase and reestablishing the pyruvate-supported liver mitochondrial capacity to handle peroxide in Nnt−/− mice.

Pyruvate Dehydrogenase Contributes to Drug Resistance of Lung Cancer Cells Through Epithelial Mesenchymal Transition.

Recently, there has been a growing interest on the role of mitochondria in metastatic cascade. Several reports have shown the preferential utilization of glycolytic pathway instead of mitochondrial respiration for energy production and the pyruvate dehydrogenase (PDH) has been considered to be a contributor to this switch in some cancers. Since epithelial mesenchymal transition (EMT) is proposed to be one of the significant mediators of metastasis, the molecular connections between cancer cell metabolism and EMT may reveal underlying mechanisms and improve our understanding on metastasis. In order to explore a potential role for PDH inhibition on EMT and associated drug resistance, we took both pharmacological and genetic approaches, and selectively inhibited or knocked down PDHA1 by using Cpi613 and shPDHA1, respectively. We found that both approaches triggered morphological changes and characteristics of EMT (increase in mesenchymal markers). This change was accompanied by enhanced wound healing and an increase in migration. Interestingly, cells were more resistant to many of the clinically used chemotherapeutics following PDH inhibition or PDHA1 knockdown. Furthermore, the TGFβRI (known as a major inducer of the EMT) inhibitor (SB-431542) together with the PDHi, was effective in reversing EMT. In conclusion, interfering with PDH induced EMT, and more importantly resulted in chemoresistance. Therefore, our study demonstrates the need for careful consideration of PDH-targeting approaches in cancer treatment.

Synthesis of pyrrothiamine, a novel thiamine analogue, and evaluation of derivatives as potent and selective inhibitors of pyruvate dehydrogenase.

Inhibition of thiamine pyrophosphate (TPP)-dependent enzymes with thiamine/TPP analogues that have the central thiazolium ring replaced with other rings is well established, but a limited number of central rings have been reported. We report a novel analogue, pyrrothiamine, with a central pyrrole ring. We further develop pyrrothiamine derivatives as potent and selective inhibitors of pyruvate dehydrogenase, which might have anti-cancer potential.

Arsenic Biotransformation: It is a complex process

Arsenic Biotransformation: It is a complex process

Abstract 10577: Dapagliflozin Improves Cardiac Function in Mice After Myocardial Infarction

Objective: We examined the effects of SGLT2 inhibition with Dapagliflozin (DAPA) on left ventricular ejection fraction (LVEF) and cardiomyocyte regeneration in mice after myocardial infraction (MI), a model for Heart Failure with reduced EF. Introduction: Neonatal mice can regenerate cardiomyocytes, but this ability is lost shortly after birth due in part to a switch from anaerobic glycolysis to fatty acid utilization. This switch is due to pyruvate dehydrogenase kinase 4 (PDK-4) inhibition of pyruvate dehydrogenase (PDH) dependent glycolysis. Empagliflozin use in mice after MI increases PDH activity and promotes glucose utilization. Hypothesis: DAPA use in mice after MI can improve LVEF by promoting cardiomyocyte regeneration secondary to increase PDH activity. Methods: A total of 49 mice, 8 weeks old, were randomized to LAD ligation (MI) or sham surgery (Sham). 72 hours after surgery, MI (n=19) and Sham (n=21) mice were randomized to PO DAPA (0.02 mg/ml) (MI-DAPA, Sham-DAPA) or vehicle (water) (MI-VEH, Sham-VEH). 20 mice were euthanized after each successive week (wk.) of treatment. Immunofluorescence, using Anti-phospho-Histone H3 (mitosis marker) (PH3)-Anti Cardiac Troponin T co-staining, was done at 2 wks. to assess regeneration. An echocardiogram was done at baseline, 72 hours after surgery, and weekly after treatment to assess LVEF. Results: At 1 wk. after MI, no significant change was observed between groups. At 2 wks., DAPA increased LVEF in MI mice (Sham-VEH 66.75%, SEM 1.18; Sham-DAPA 78.63%, SEM 2.82; MI-VEH 56.86%, SEM 4.80; MI-DAPA 74.3%, SEM 0.58%, p<0.05). At 2 wks., MI increased % of PH3 positive cardiomyocyte nuclei (Sham-VEH 0%, SEM 0; MI-VEH 0.4268%, SEM 0.1259; p<0.05). In DAPA treated mice no significant difference was observed as the % of PH3 positive cardiomyocyte increased in Sham-DAPA mice (Sham-DAPA 0.2253%, SEM 0.078; MI-DAPA 0.46%, SEM 0.07; p 0.47). Despite an increase in SHAM-VEH mice, no statistically significant difference was observed between SHAM-VEH and SHAM-DAPA. Conclusions: DAPA mitigate cardiac remodeling in mice after MI with increase LVEF after 2 wks. MI increases mitosis in cardiomyocytes after two wks. Despite a trend for increased mitosis with DAPA treatment for 2 wks., no significant change was seen.

Abstract PO-024: Targeting cellular metabolism with CPI-613 sensitizes pancreatic cancer cells to radiotherapy

Abstract Novel treatment strategies for pancreatic ductal adenocarcinoma (PDAC) are desperately needed. Local tumor progression is a cause of significant morbidity and mortality in patients with surgically unresectable disease. Often, regional anatomic structures limit the total doses of radiation therapy (RT) that can be safely delivered. Conventional doses of concurrent chemotherapy and RT (chemo-RT) have shown suboptimal results in local control of disease, progression free survival, and overall survival. Therefore, novel and effective approaches to enhance the efficacy of RT are urgently needed to improve overall survival in unresectable PDAC. Metabolic reprogramming enables cancer cells to adjust their metabolism to support increased energy requirements associated with continuous growth and proliferation. Indeed, metabolic reprogramming is a hallmark of PDAC and is associated with increased tumor cell plasticity and chemo-RT resistance. Cancer-cell mitochondria are key regulators of deranged tumor metabolism and have been shown to guide molecular pathways involved in radio-resistance. There is also expanding data that the presence of metabolites modulates the response of cancer cells to RT primarily by impacting the ability to repair DNA. This makes them an optimal candidate for novel radiosensitization strategies, as these characteristics are unique to PDAC cells, and are limited in normal cells. CPI-613, is an analog of lipoic acid which inhibits pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (α-KGDH), thereby disrupting mitochondrial metabolism leading to selective tumor cell killing. The drug has demonstrated significant clinical activity in patients with metastatic PDAC in combination with standard of care chemotherapies. It remains unknown as to the efficacy in patients treated with concurrent chemo-RT. Here we show that combined treatment of RT (2 and 10 Gy) with CPI-613 (used at 200 and 300 μM) causes superior inhibition of pancreatic cancer cell growth (MTT assay and colony formation assay). In addition, we demonstrate enhanced apoptosis (Annexin V FITC and 7AAD assay) of PDAC cells when treated with a combination of RT and CPI-613. Molecular analysis revealed superior inhibition of PDH and α-KGDH at the protein level. Targeted metabolomic analysis on PDAC cells post CPI-613-RT treatment revealed alterations in key mitochondrial metabolites, leading to these findings. These results indicate broader target engagement by this combination treatment, indicating the sensitization of CPI-613 treated PDAC cells to radiotherapy at doses as low as 2 Gy. Furthermore, in our preclinical cellular models, a combination treatment of CPI-613 with either Gemcitabine or 5-FU has shown synergistic effects on the proliferation of PDAC cells. Pre-clinical anti-tumor efficacy of the CPI-613-RT and CPI-613-RT-chemo using subcutaneous and orthotopic PDAC models is planned. Our results bring forward a novel combination of CPI-613-RT that warrants further pre-clinical and early phase clinical investigations. Citation Format: William A. Hall, Husain Y. Khan, Mandana Kamgar, Susan Tsai, Kathleen Christians, Douglas B. Evans, Philip Philip, Callisia Clarke, Ben George, Beth Erickson, Asfar S. Azmi. Targeting cellular metabolism with CPI-613 sensitizes pancreatic cancer cells to radiotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-024.

DCA Protects against Oxidation Injury Attributed to Cerebral Ischemia-Reperfusion by Regulating Glycolysis through PDK2-PDH-Nrf2 Axis.

Cerebral ischemic stroke (IS) is still a difficult problem to be solved; energy metabolism failure is one of the main factors causing mitochondrion dysfunction and oxidation stress damage within the pathogenesis of cerebral ischemia, which produces considerable reactive oxygen species (ROS) and opens the blood-brain barrier. Dichloroacetic acid (DCA) can inhibit pyruvate dehydrogenase kinase (PDK). Moreover, DCA has been indicated with the capability of increasing mitochondrial pyruvate uptake and promoting oxidation of glucose in the course of glycolysis, thereby improving the activity of pyruvate dehydrogenase (PDH). As a result, pyruvate flow is promoted into the tricarboxylic acid cycle to expedite ATP production. DCA has a protective effect on IS and brain ischemia/reperfusion (I/R) injury, but the specific mechanism remains unclear. This study adopted a transient middle cerebral artery occlusion (MCAO) mouse model for simulating IS and I/R injury in mice. We investigated the mechanism by which DCA regulates glycolysis and protects the oxidative damage induced by I/R injury through the PDK2-PDH-Nrf2 axis. As indicated from the results of this study, DCA may improve glycolysis, reduce oxidative stress and neuronal death, damage the blood-brain barrier, and promote the recovery of oxidative metabolism through inhibiting PDK2 and activating PDH. Additionally, DCA noticeably elevated the neurological score and reduced the infarct volume, brain water content, and necrotic neurons. Moreover, as suggested from the results, DCA elevated the content of Nrf2 as well as HO-1, i.e., the downstream antioxidant proteins pertaining to Nrf2, while decreasing the damage of BBB and the degradation of tight junction proteins. To simulate the condition of hypoxia and ischemia in vitro, HBMEC cells received exposure to transient oxygen and glucose deprivation (OGD). The DCA treatment is capable of reducing the oxidative stress and blood-brain barrier of HBMEC cells after in vitro hypoxia and reperfusion (H/R). Furthermore, this study evidenced that HBMEC cells could exhibit higher susceptibility to H/R-induced oxidative stress after ML385 application, the specific inhibitor of Nrf2. Besides, the protection mediated by DCA disappeared after ML385 application. To sum up, as revealed from the mentioned results, DCA could exert the neuroprotective effect on oxidative stress and blood-brain barrier after brain I/R injury via PDK2-PDH-Nrf2 pathway activation. Accordingly, the PDK2-PDH-Nrf2 pathway may play a key role and provide a new pharmacology target in cerebral IS and I/R protection by DCA.

Calcineurin inactivation inhibits pyruvate dehydrogenase complex activity and induces the Warburg effect.

Calcineurin is a calcium- and calmodulin-dependent serine/threonine protein phosphatase that connects the Ca2+-dependent signalling to multiple cellular responses. Calcineurin inhibitors (CNIs) have been widely used to suppress immune response in allograft patients. However, CNIs significantly increase cancer incidence in transplant recipients compared with the general population. Accumulating evidence suggests that CNIs may promote the malignant transformation of cancer cells in addition to its role in immunosuppression, but the underlying mechanisms remain poorly understood. Here, we show that calcineurin interacts with pyruvate dehydrogenase complex (PDC), a mitochondrial gatekeeper enzyme that connects two key metabolic pathways of cells, glycolysis and the tricarboxylic acid cycle. Mitochondrial-localized calcineurin dephosphorylates PDHA1 at Ser232, Ser293 and Ser300, and thus enhances PDC enzymatic activity, remodels cellular glycolysis and oxidative phosphorylation, and suppresses cancer cell proliferation. Hypoxia attenuates mitochondrial translocation of calcineurin to promote PDC inactivation. Moreover, CNIs promote metabolic remodelling and the Warburg effect by blocking calcineurin-mediated PDC activation in cancer cells. Our findings indicate that calcineurin is a critical regulator of mitochondrial metabolism and suggest that CNIs may promote tumorigenesis through inhibition of the calcineurin-PDC pathway.

Protective effects of dichloroacetic acid on endometrial injury and ovarian reserve in an experimental rat model of diabetes mellitus

To study (1) ovarian and endometrial damage caused by the hyperglycemia and (2) the effects of dichloroacetic acid (DCA) on follicular reserve and endometrial damage in streptozocin induced diabetic rats. This study consisted 24 rats randomly separated into three groups. A diabetes model was achieved in 16 rats experimentally, and normoglycemic eight rats were assigned as control group (Group 1). The rats with diabetes were randomly separated to two groups: 1 mL/kg/day intraperitoneal 0.9% NaCl was given to eight rats as diabetic vehicle (Group 2) and 10mg/kg/day DCA was given to other eight rats as DCA treated group (Group 3). Hysterectomy with bilateral oophorectomy was performed for histopathological evaluation and blood samples were collected after 4 weeks. Diabetes caused ovarian and endometrial damage (p < 0.0001). Pentraxin-3 (PTX-3), lactic acid, and transforming growth factor-beta (TGF-β) were higher (p < 0.05, p < 0.05, and p < 0.0001, respectively), whereas anti-Mullerian hormone (AMH) was lower in diabetic rats (p < 0.05). These findings reflected the diabetic damage in the genital tract and diminished ovarian reserve occurred via fibrosis, severe inflammation, and oxidative stress. DCA improved the histopathological fibrosis and degeneration in the ovaries and endometrium (p < 0.05). There was a concominant decrease of TGF-β and lactic acid levels with DCA treatment (p < 0.05). DCA also improved ovarian reserve with higher AMH levels (p < 0.05). The several unfavored changes in the endometrium and ovaries due to diabetes have been determined in this present study. DCA might provide the continuity of the endometrial cycle, physiological endometrial structure, ovarian follicular growth, oocyte maturation, and physiological ovarian function by decreasing the lactate levels via inhibiting pyruvate dehydrogenase kinase enzyme.

Targeting Glucose Metabolism of Cancer Cells with Dichloroacetate to Radiosensitize High-Grade Gliomas.

As the cornerstone of high-grade glioma (HGG) treatment, radiotherapy temporarily controls tumor cells via inducing oxidative stress and subsequent DNA breaks. However, almost all HGGs recur within months. Therefore, it is important to understand the underlying mechanisms of radioresistance, so that novel strategies can be developed to improve the effectiveness of radiotherapy. While currently poorly understood, radioresistance appears to be predominantly driven by altered metabolism and hypoxia. Glucose is a central macronutrient, and its metabolism is rewired in HGG cells, increasing glycolytic flux to produce energy and essential metabolic intermediates, known as the Warburg effect. This altered metabolism in HGG cells not only supports cell proliferation and invasiveness, but it also contributes significantly to radioresistance. Several metabolic drugs have been used as a novel approach to improve the radiosensitivity of HGGs, including dichloroacetate (DCA), a small molecule used to treat children with congenital mitochondrial disorders. DCA reverses the Warburg effect by inhibiting pyruvate dehydrogenase kinases, which subsequently activates mitochondrial oxidative phosphorylation at the expense of glycolysis. This effect is thought to block the growth advantage of HGGs and improve the radiosensitivity of HGG cells. This review highlights the main features of altered glucose metabolism in HGG cells as a contributor to radioresistance and describes the mechanism of action of DCA. Furthermore, we will summarize recent advances in DCA’s pre-clinical and clinical studies as a radiosensitizer and address how these scientific findings can be translated into clinical practice to improve the management of HGG patients.

P-176 A multicenter, randomized phase 1b/2 study of gemcitabine and cisplatin with or without CPI-613 as first-line therapy for patients with advanced unresectable biliary tract cancer (BilT-04)

Patients (pts) with advanced biliary tract cancers (BTC) have poor prognosis despite systemic chemotherapy. Gemcitabine and cisplatin is a standard first-line systemic therapy with an overall response rate (ORR) of 26% and a median overall survival of 11.7 months. This investigator-initiated, multi-institutional phase 1b/2 trial is designed to investigate the role of gemcitabine, cisplatin, and CPI-613 in pts with advanced BTC. CPI-613 is a stable intermediate of a lipoate analog that inhibits pyruvate dehydrogenase and a-ketoglutarate dehydrogenase enzymes of the tricarboxylic (TCA) cycle preferentially within the mitochondria of cancer cells.

Interim Analysis of a Single Arm Phase II Study of Cpi-613 in Combination with High Dose Cytarabine and Mitoxantrone for Patients with Relapsed or Refractory AML

Background:CPI-613 is a first in class agent that inhibits pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase. We have previously shown CPI-613 inhibits mitochondrial respiration, causes phosphorylation of PDH, activation of adenosine monophosphate activated kinase in acute myeloid leukemia (AML) cells. We also showed that CPI-163 could be safely combined with high dose cytarabine (HiDAC) and mitoxantrone in patients with relapsed or refractory AML. Early efficacy data suggested that this combination increased the response rate and prolonged survival in elderly patients when compared to historical data.Methods:We opened a Phase II trial of CPI-613 in combination with HiDAC and mitoxantrone for relapsed or refractory AML patients to confirm the safety and efficacy seen in the Phase I trial, as well as to expand several dosing cohorts. In the current trial, patients are given CPI-613 as a 2-hour infusion on days 1 through 5. HiDAC is dosed at 3,000 mg/m² (if younger than 60) or at 1,500 mg/m² (if 60 years of age or older), given every 12 hours for 5 doses, starting on day 3 following the CPI-613 infusion. The mitoxantrone was dosed at 6 mg/m² and is given once daily following the first, third and fifth HiDAC doses. At day 14, nadir marrow was evaluated and, if significant residual leukemia was present, patients could be re-treated as above or with an abbreviated 3-day cycle. Nadir marrow was re-evaluated in patients receiving a second cycle, and those with residual disease at that point were removed from study and considered refractory. Responding patients were eligible to receive up to 2 abbreviated 3-day consolidation cycles. Patients who could not go on to allogeneic stem cell transplant could receive maintenance therapy consisting of CPI-613 at 2,500 mg/m², given on days 1 through 5 every 28 days. The first 7 patients were enrolled at the maximum tolerated dose of CPI-613 2,500 mg/m2, the next 17 patients were enrolled at a dose of 2,000 mg/m2 and 6 patients have been enrolled in the current 1,500 mg/m2 cohort.Results:At the time of this interim analysis, 30 patients are evaluable. The median age is 64 years (range 21-78). Disease was relapsed in 77% of patients (23/30) and refractory in in 23% (7/30). In patients with relapsed disease, the median duration of first complete response (CR) was 12 months. In 80% of patients, this was their first salvage regimen. AML was secondary in 5 patients, 4 with prior MDS and 1 with prior atypical chronic myeloid leukemia. Cytogenetics were poor risk in 12 patients, intermediate in 16, and good in 2. In the intention to treat (IIT) cohort (n=30) the overall response rate was 53% (10 CR + 5 CR with incomplete count recovery + 1 partial response), with a median survival of 10.4 months (Figure 1A). Response was significantly associated with survival, with a median survival of 12.5 months in responders compared to 1.6 months for all others (Figure 1B). In patients ≥60 years old, the response rate was 48% (10/21) with a median survival of 10 months. Consistent with the Phase I results, this was not significantly different from patients younger than 60 years old (p=0.6345, Figure 1C). After hematological and infectious, the most common clinically significant Grade 3 or 4 toxicities of the combination were diarrhea and anorexia. The 30-day mortality rate was 7% (2/30). Seven patients (23%) went on to allogeneic stem cell transplantation, 5 of whom were 60 years of age or older. Mutational status for FLT3, NPM, or CEBPa was available for all patients enrolled. Additional mutational testing was available for 57% of patients. In an exploratory analysis of recurring mutations, we found that 6 of 7 patients (86%) with a DNMT3a mutation achieved a response, as did all 8 patients (100%) with an NPM1 mutation (Table 1).Conclusions: TCA cycle inhibition with CPI-613 is a promising novel approach in the treatment of AML patients, especially in the elderly and those with NPM1 and DNMT3a mutations.Figure 1. Kaplan-Meier analyses of overall survival (OS) in: the entire IIT cohort (A), 60 years of age and older vs younger than 60 (B) and Responders vs No response (C). CHAM, combined high-dose AraC (cytarabine) and mitoxantrone; CR, complete remission; CRi, complete remission with incomplete count recovery; PR, partial remission. [Display omitted] DisclosuresPardee:Rafael Pharmaceuticals: Employment, Research Funding. Ellis:Alexion: Speakers Bureau. Powell:Rafael Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Pyruvate Dehydrogenase (PDH) Activity Is a Source of Resistance to Sorafenib in FLT3-ITD Expressing Acute Myeloid Leukemia (AML)

Background:AML is characterized by poor prognoses and resistance to therapy. Despite decades of research long term survival remains 30-40%, and little is known about the mechanisms involved in resistance. Cells that are resistant to cytarabine have increased oxidative metabolism and mitochondrial mass, thus suggesting a role of mitochondrial metabolism in resistance to therapy. Our lab has previously shown that genetic and pharmacologic inhibition of pyruvate dehydrogenase (PDH) sensitizes AML cells to DNA damaging agents, specifically cytarabine and doxorubicin. CPI-613 is a first in class agent that inhibits PDH and has been shown to sensitize AML cells to DNA damaging agents. How AML cells alter their mitochondrial metabolism in response to targeted therapies is not well known. Sorafenib is a tyrosine kinase inhibitor that has been shown to inhibit FLT3. Activating mutations in FLT3 are found in about 30% of AML patients with the most common mutation being an internal tandem duplication (FLT3-ITD). Sorafenib has limited activity as a single agent against FLT3 mutated AML. The degree to which mitochondrial metabolism contributes to resistance to sorafenib is currently unknown.Methods:A murine AML cell line expressing the FLT3-ITD (MFL2) was used to knockout PDH through the CRISPR-Cas9 system, and the safe-harbor ROSA26 locus was used as control. Knockout of PDH was confirmed by Western blot analysis. PDH knockout cells were treated with sorafenib at different doses for 24 and 72 hours. Viability assays were done using the EZQuant assay. Mitochondrial mass was assessed on PDH KO and ROSA26 control cells that were treated with sorafenib after 24-hours by staining the cells with Mitotracker and measuring uptake by flow cytometry. Apoptosis was assessed through annexin V and Propidium Iodide staining. Oxygen consumption was assessed using the Seahorse XF24 Extracellular Flux Analyzer. MOLM-13 (human AML cell line that expresses the FLT3-ITD) cells were treated with sorafenib with and without CPI-613 and combinatorial index values determined by the method of Chou and Talalay.Results:PDH deleted cells (PDH KO) were significantly more sensitive to sorafenib than ROSA control cells at several doses (p value <0.0001). This sensitivity is in part due to increased apoptosis, which was shown by a significant increase of Annexin V positive PDH KO cells compared to controls (p value <0.001). Interestingly, when mitochondrial oxygen consumption rates (OCR) were assessed after 24-hour sorafenib exposure, ROSA controls showed decreased OCR implying a direct effect on mitochondrial function. PDH KO cells showed low basal mitochondrial OCR that did not change with treatment of sorafenib, consistent with previous data. Mitochondrial mass was also assessed and ROSA controls showed an increase of mitochondrial mass with treatment of sorafenib at 24-hours while PDH KO cells showed no increase. CPI-613 synergized with sorafenib in MOLM-13 cells with a combinatorial index value of 0.581 (95% CI 0.4520-0.7107).Conclusion:Here we show that genetic deletion of PDH sensitizes FLT3-ITD positive cells to sorafenib. Mechanistically, sorafenib inhibits mitochondrial function as indicated by ROSA cells showing decreased OCR while increasing mitochondrial mass. PDH KO cells lack the mitochondrial reserve to counteract this affect resulting in increased cell death. A FLT3-ITD expressing cell line could be sensitized to sorafenib treatment by exposure to the TCA cycle inhibitor CPI-613. Considered in conjunction with previous findings, these results suggest that the PDH activity is an important factor in developing resistance to multiple classes of targeted and chemotherapeutic agents and suggests that CPI-613 could increase responses to targeted agents in AML. DisclosuresPardee:Rafael Pharmaceuticals: Employment, Research Funding.

Glutamine Catabolism Maintains MCL-1 Expression in Glucose-Deprived Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy accounting for 11,000 deaths annually in the US with the majority succumbing to disease due to the development of resistance. A key component to the development of resistance lies in ineffective engagement of the BCL-2 family of apoptosis regulators. We and others have demonstrated that glucose maintains levels of a key resistance-promoting BCL-2 family member, myeloid cell leukemia factor 1 (MCL-1). Expression of MCL-1 promotes resistance of MM to a wide range of therapeutics. MM, like other cancers, exhibits elevated glucose consumption associated with manifestation of the Warburg effect. We previously reported that myeloma cells maintain the glycolytic phenotype by achieving constitutive plasma membrane localization of GLUT4, and GLUT4 inhibition or glucose deprivation suppressed MCL-1 expression. In this study, we further investigated the mechanism underlying the suppression of MCL-1 expression upon glucose deprivation.L363 and KMS18 cell lines are sensitive to glucose deprivation, exhibiting significant cytotoxicity upon glucose deprivation and demonstrating rapid reduction of MCL-1 protein in contrast to JJN3 and KMS11 cells that are resistant to glucose deprivation. Knockdown of MCL-1 in JJN3 dramatically induced apoptosis while overexpression of MCL-1 in L363 significantly reversed cell death upon glucose deprivation, supporting a role for MCL-1 suppression in mediating glucose-deprivation elicited cell death. We next proceeded to investigate the metabolic pathways contributing to the persistence of MCL-1 in the glucose-insensitive lines. Since glutamine is an important carbon source that can compensate for glucose deprivation, we examined glutamine uptake in L363 and JJN3 cells. Evaluation of 3H-glutamine uptake revealed that JJN3 cells exhibit higher glutamine uptake than L363 cells. In addition, we performed 13C-based metabolic flux analysis in order to identify metabolites that may be contributing to the maintenance of MCL-1 in glucose-deprived cells. Interestingly the level of glutathione (GSH) was much higher in JJN3 than in L363 that was maintained even upon glucose deprivation. Since GSH is a major cellular reactive oxygen species (ROS) scavenger, we evaluated levels of ROS using a cyto-roGFP construct and found that it was significantly elevated upon glucose withdrawal in L363 but not in JJN3. In addition, treating glucose deprived L363 with cell-permeant GSH monoethyl ester (GSH-MEE) rescued MCL-1 expression, further supporting a role for ROS in suppressing MCL-1 expression.Glutamine uptake evaluation revealed that glutamine consumption was maintained upon glucose withdrawal in both JJN3 and L363, so we were then interested in the TCA cycle-related differences in glutamine metabolism between JJN3 and L363. The level of pyruvate dehydrogenase alpha 1 (PDHA1) phosphorylation decreased upon glucose deprivation only in JJN3 cells, which activates PDHA1 and allows pyruvate to flow into the TCA cycle. In accordance with this, treatment of JJN3 with dichloroacetate (DCA), which inhibits pyruvate dehydrogenase kinase and therefore activates PDHA1, restored MCL-1 expression and prevented cell death of L363 during glucose deprivation. To study how TCA cycle helps maintain the GSH level, glutamate dehydrogenase (GDH) knockdown cells were generated because GDH catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and glutamate is the precursor for GSH. Interestingly, in GDH knockdown L363 cells, treatment of DCA was not able to reverse MCL-1 protein level upon glucose deprivation. Taken together, our observations suggest that glucose-deprived resistant cells sustain higher levels of GSH via greater glutamine uptake, thereby scavenging ROS to sustain cell viability; while sensitive cells cannot make GSH effectively, leading to increased ROS level, decreased MCL-1 expression, and ultimately, apoptosis.In sum, we have discovered that targeting glucose entry elicits apoptosis via an induction of ROS leading to suppression of MCL-1. These observations provide further rationale for the development of GLUT4-specific biologics to target aberrant glucose metabolism in myeloma and the need to further understand how glutamine metabolism and the TCA cycle contribute to maintaining MCL-1 expression. DisclosuresNo relevant conflicts of interest to declare.

Selenium (Se) as well as mercury (Hg) may influence the methylation and toxicity of inorganic arsenic, but further research is needed with combination of Inorg-arsenic, Se, and Hg

Selenium (Se) as well as mercury (Hg) may influence the methylation and toxicity of inorganic arsenic, but further research is needed with combination of Inorg-arsenic, Se, and Hg

Identification of PDHX as a metabolic target for esophageal squamous cell carcinoma.

The metabolism in tumors is reprogrammed to meet its energetic and substrate demands. However, this metabolic reprogramming creates metabolic vulnerabilities, providing new opportunities for cancer therapy. Metabolic vulnerability as a therapeutic target in esophageal squamous cell carcinoma (ESCC) has not been adequately clarified. Here, we identified pyruvate dehydrogenase (PDH) component X (PDHX) as a metabolically essential gene for the cell growth of ESCC. PDHX expression was required for the maintenance of PDH activity and the production of ATP, and its knockdown inhibited the proliferation of cancer stem cells (CSCs) and in vivo tumor growth. PDHX was concurrently upregulated with the CD44 gene, a marker of CSCs, by co‐amplification at 11p13 in ESCC tumors and these genes coordinately functioned in cancer stemness. Furthermore, CPI‐613, a PDH inhibitor, inhibited the proliferation of CSCs in vitro and the growth of ESCC xenograft tumors in vivo. Thus, our study provides new insights related to the development of novel therapeutic strategies for ESCC by targeting the PDH complex‐associated metabolic vulnerability.

A multicenter, randomized phase 1b/2 study of gemcitabine and cisplatin with or without CPI-613 as first-line therapy for patients with advanced unresectable biliary tract cancer (BilT-04).

TPS4158 Background: Patients (pts) with advanced biliary tract cancers (BTC) have poor prognosis despite systemic chemotherapy. Gemcitabine and cisplatin is a standard first-line systemic therapy with an overall response rate (ORR) of 26% and a median overall survival of 11.7 months. This investigator-initiated, multi-institutional phase 1b/2 trial is designed to investigate the role of gemcitabine, cisplatin and CPI-613 in pts with advanced BTC. CPI-613 is a stable intermediate of a lipoate analog that inhibits pyruvate dehydrogenase and a-ketoglutarate dehydrogenase enzymes of the tricarboxylic (TCA) cycle preferentially within the mitochondria of cancer cells. Methods: Key eligibility criteria include histologically confirmed, metastatic or unresectable BTC (intra- or extra-hepatic and gallbladder) without prior systemic treatment, measurable disease per RECIST v1.1, and ECOG PS 0-1. Primary objective of the phase 1b portion (n = 20 pts; TiTE-CRM methodology) is to determine the recommended phase 2 dose of the combination, and for the phase 2 portion, ORR (n = 48-58 pts; 2:1 randomization). Assuming a null hypothesis ORR of 25% and an alternative hypothesis of 43%, this ongoing trial has at least 80% power with a one-sided alpha of 0.05 to identify treatment efficacy of the study arm. Secondary objectives include evaluation of progression-free survival, overall survival, and safety in this patient population. Exploratory objectives include identification of molecular markers of response and resistance in tumor samples and serially collected blood (pre-, on-, and post-therapy), including whole exome/transcriptomic analysis, and immunohistochemical staining (PDK, PDH, KGDH, SOD2 and CD79a). Gemcitabine 1000 mg/m2, cisplatin 25 mg/m2 with or without CPI-613 (dose levels: 500 mg/m2, 1000 mg/m2, 1500 mg/m2, and 2000 mg/m2) will be given IV on days 1 and 8 every 21 days. In the absence of disease progression, pts may continue therapy for up to 2 years. Total accrual goal is 68-78 evaluable pts. To date, 5 of planned 20 pts enrolled on the phase 1b portion are without dose limiting toxicity. Clinical trial information: NCT04203160.

A phase II open-label study of cpi-613 in combination with modified (m)FOLFIRINOX in patients with locally advanced pancreatic cancer.

TPS4176 Background: For patients with locally advanced pancreatic cancer, neoadjuvant trials are the preferred strategy. The goals of neoadjuvant treatment are to diminish the size of the primary tumor to allow for safe surgical resection and to limit the chance of developing metastatic disease. mFOLFIRINOX is the gold-standard for treatment in the adjuvant setting and an acceptable regimen in the neoadjuvant setting with many ongoing neoadjuvant trials using it as a chemotherapeutic backbone. CPI-613 (devimistat) is a small-molecule inhibitor of pyruvate dehydrogenase and alpha-ketogluterate dehydrogenase that has been studied in combination with mFOLFIRINOX in a phase I trial of patients with metastatic pancreas cancer and shown to be safe at the proposed phase II dose. Methods: This is a single-center, single-arm phase II trial for patients with locally advanced pancreatic cancer; defined as either borderline resectable or unresectable according to NCCN guidelines and interpreted by the primary investigator. Patients with metastatic disease are excluded. Patients will receive treatment with CPI-613 and mFOLFIRINOX per the table below. The primary endpoint is overall survival. Secondary endpoints are progression free survival and resection rate. At the time of submission this study has completed initial accrual with 37 patients enrolled. Clinical trial information: NCT03699319. [Table: see text]