Tumors frequently display a glycolytic phenotype with increased flux through glycolysis and concomitant synthesis of lactate. To maintain glycolytic flux and prevent intracellular acidification, tumors efflux lactate via lactate transporters (MCT1-4). Inhibitors of lactate transport have the potential to inhibit glycolysis and tumor growth. We developed a small molecule inhibitor of MCT1 (AZD3965) and assessed its activity across a panel of cell lines. We explored its antitumor activity as monotherapy and in combination with doxorubicin or rituximab. AZD3965 is a potent inhibitor of MCT1 with activity against MCT2 but selectivity over MCT3 and MCT4. In vitro, AZD3965 inhibited the growth of a range of cell lines especially haematological cells. Inhibition of MCT1 by AZD3965 inhibited lactate efflux and resulted in accumulation of glycolytic intermediates. In vivo, AZD3965 caused lactate accumulation in the Raji Burkitt’s lymphoma model and significant tumor growth inhibition. Moreover, AZD3965 can be combined with doxorubicin or rituximab, components of the R-CHOP standard-of-care in DLBCL and Burkitt’s lymphoma. Finally, combining lactate transport inhibition by AZD3965 with GLS1 inhibition in vitro, enhanced cell growth inhibition and cell death compared to monotherapy treatment. The ability to combine AZD3965 with novel, and standard-of-care inhibitors offers novel combination opportunities in haematological cancers.
Tumors frequently display a glycolytic phenotype with increased flux through glycolysis and concomitant synthesis of lactate. To maintain glycolytic flux and prevent intracellular acidification, tumors efflux lactate via lactate transporters (MCT1-4). Inhibitors of lactate transport have the potential to inhibit glycolysis and tumor growth. We developed a small molecule inhibitor of MCT1 (AZD3965) and assessed its activity across a panel of cell lines. We explored its antitumor activity as monotherapy and in combination with doxorubicin or rituximab. AZD3965 is a potent inhibitor of MCT1 with activity against MCT2 but selectivity over MCT3 and MCT4. In vitro, AZD3965 inhibited the growth of a range of cell lines especially haematological cells. Inhibition of MCT1 by AZD3965 inhibited lactate efflux and resulted in accumulation of glycolytic intermediates. In vivo, AZD3965 caused lactate accumulation in the Raji Burkitt's lymphoma model and significant tumor growth inhibition. Moreover, AZD3965 can be combined with doxorubicin or rituximab, components of the R-CHOP standard-of-care in DLBCL and Burkitt's lymphoma. Finally, combining lactate transport inhibition by AZD3965 with GLS1 inhibition in vitro, enhanced cell growth inhibition and cell death compared to monotherapy treatment. The ability to combine AZD3965 with novel, and standard-of-care inhibitors offers novel combination opportunities in haematological cancers.
Due to increased reliance on glycolysis, which produces lactate, monocarboxylate transporters (MCTs) are often upregulated in cancer. MCT4 is associated with the export of lactic acid from cancer cells under hypoxia, so inhibition of MCT4 may lead to cytotoxic levels of intracellular lactate. In addition, tumorderived lactate is known to be immunosuppressive, so MCT4 inhibition may be of interest for immuno-oncology. At the outset, no potent and selective MCT4 inhibitors had been reported, but a screen identified a triazolopyrimidine hit, with no close structural analogues. Minor modifications to the triazolopyrimidine were made, alongside design of a constrained linker and broad SAR exploration of the biaryl tail to improve potency, physical properties, PK, and hERG. The resulting clinical candidate 15 (AZD0095) has excellent potency (1.3 nM), MCT1 selectivity (>1000×), secondary pharmacology, clean mechanism of action, suitable properties for oral administration in the clinic, and good preclinical efficacy in combination with cediranib.
Metabolic adaptation to a Warburg phenotype is a hallmark of cancer, where tumors rely on glucose to produce ATP and building blocks required to support rapid tumor growth. Glycolytic metabolism requires continuous export of lactate from cells by monocarboxylate transporters (MCTs) resulting in accumulation of lactic acid in the tumor microenvironment. Tumor-derived lactate is immune-suppressive and has been shown to modify immune cell function. Inhibition of the MCT4 lactate transporter has the potential to reduce lactate in the tumor microenvironment and reverse immune suppression. AZD0095 is a highly selective, potent inhibitor of MCT4 with a cellular activity of 1-3 nM with a >1000-fold selectivity over MCT1. We confirm that lactic acid inhibits T cell proliferation and viability. AZD0095 does not inhibit T cell proliferation or survival (IC50 > 10 μM). Oral administration of AZD0095 (10-100 mg/kg BID) modulates lactate transport in mouse syngeneic models which do not express the MCT1 lactate transporter. AZD0095 treatment causes an increase in tumor-infiltrating lymphocytes and an increase in cytotoxic T cells in combination with α-PD1. AZD0095 treatment suppresses myeloid infiltration as a monotherapy and in combination with checkpoint inhibitor. Consistent with the observed changes in immune cell infiltration, the combination of AZD0095 with checkpoint inhibitors α-PD1 or α-CTLA4 (10 mg/kg 3x week) enhances the anti-tumor activity of checkpoint inhibitor monotherapy in MC-38 and EMT6 (MCT1 knock-out) mouse syngeneic models. These data demonstrate that AZD0095 can reverse lactate-driven immunosuppression and enhance response to checkpoint inhibition in pre-clinical models. Citation Format: Susan E. Critchlow, Gareth Hughes, Anna Staniszewska, Matt King, Filippos Michopoulos, Lorna Hopcroft, Martin R. Brown, Jenna Bradley, Beverley Hammond, Pablo MorentinGutierrez, Larissa Carnevalli, Elizabeth Hardaker, Frederick W. Goldberg. Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1207.
Lactic acid transport is a key process maintaining glycolytic flux in tumors. Inhibition of this process will result in glycolytic shutdown, impacting on cell growth and survival and thus has been pursued as a therapeutic approach for cancers. Using a cell-based screen in a MCT4-dependent cell line, we identified and optimized compounds for their ability to inhibit the efflux of intracellular lactic acid with good physical and pharmacokinetic properties. To deconvolute the mechanism of lactic acid efflux inhibition, we have developed three assays to measure cellular target engagement. Specifically, we synthesized a biologically active photoaffinity probe (IC50 < 10 nM), and using this probe, we demonstrated selective engagement of MCT4 of our parent molecule through a combination of confocal microscopy and in-cell chemoproteomics. As an orthogonal assay, the cellular thermal shift assay (CETSA) confirmed binding to MCT4 in the cellular system. Comparisons of lactic acid efflux potencies in cells with differential expression of MCT family members further confirmed that the optimized compounds inhibit the efflux of lactic acid through the inhibition of MCT4. Taken together, these data demonstrate the power of orthogonal chemical biology methods to determine cellular target engagement, particularly for proteins not readily amenable to traditional biophysical methods.
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