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DIPG harbour alterations targetable by MEK inhibitors, with acquired resistance mechanisms overcome by combinatorial inhibition

ABSTRACT: The survival of children with DIPG remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib in samples which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAFG469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and the patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted a confirmed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: James Wright

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD019701 | Pride | 2021-07-20

REPOSITORIES: Pride

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Project description:The mitogen-activated protein kinase (MAPK) pathway is one of the most altered pathways in cancer. It is involved in the control of cell proliferation, invasion, metabolism, and resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer and glioma, are driven by an activating missense mutation (V600E) in one component of the pathway, BRAF. BRAF V600E mutated cancers may respond initially to MEK inhibition, but may develop resistance mediated by increased reliance on mTOR signaling. We have previously demonstrated that the combination of the MEK inhibitor trametinib with the dual mTORC1/2 inhibitor TAK228 improved survival and decreased vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of, and the changes in response to, MEK and mTOR inhibition, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228, alone and in combination. We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the MAPK and mTOR pathways, increased apoptotic signaling and suppressed angiogenesis signaling. Furthermore, we found that trametinib and TAK228 combination treatment broadly suppressed the activity of the cyclin-dependent kinases and increased the levels of proteins (and their activating phosphorylations) involved in glycolysis, the TCA cycle, nucleotide biosynthesis and DNA replication. We also demonstrated activation of both receptor tyrosine kinase and histone deacetylase proteins. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies a number of targetable upregulated proteins and pathways, providing new avenues for the development of additional rational combination therapies for aggressive BRAF-driven tumors.

Project description:The first clinical trial testing the combination of targeted therapy with a BRAF inhibitor vemurafenib and immunotherapy with a CTLA-4 antibody ipilimumab was terminated early due to significant liver toxicities, possibly due to paradoxical activation of the MAPK pathway by BRAF inhibitors in tumors with wild type BRAF. MEK inhibitors can potentiate the MAPK inhibition in tumor, while potentially alleviating the unwanted paradoxical MAPK activation. With a mouse model of syngeneic BRAFV600E driven melanoma (SM1), we tested whether the addition of the MEK inhibitor trametinib would enhance the immunosensitization effects of the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression. Bioluminescent imaging and tumor infiltrating lymphocyte (TIL) phenotyping showed increased effector infiltration to tumors with dabrafenib, trametinib or dabrafenib plus trametinib with pmel-1 ACT combination. Intracellular IFN gamma staining of the TILs and in vivo cytotoxicity studies showed trametinib was not detrimental to the effector functions in vivo. Dabrafenib increased tumor associated macrophages and T regulatory cells (Tregs) in the tumors, which can be overcome by addition of trametinib. Microarray analysis revealed increased melanoma antigen, MHC expression, and global immune-related gene upregulation with the triple combination therapy. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen specific ACT, we tested the triple combination of dabrafenib, trametinib with anti-PD1 therapy, and observed superior anti-tumor effect to SM1 tumors. Our findings support the testing of these combinations in patients with BRAFV600E mutant metastatic melanoma. SM1 tumors were implanted into C57BL/6 mice. Mice were treated by ACT of pmel-1 splenocytes or C57BL/6 splenocytes as control. Pmel-1 treated mice were additionally treated with either vehicle, dabrafenib, trametinib, or combination of both drugs and control mice were treated with vehicle or combination of both drugs.

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DIPG harbour alterations targetable by MEK inhibitors, with acquired resistance mechanisms overcome by combinatorial inhibition

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