Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

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Supplementary Figure S3. JIB-04 inhibits translation initiation.

Effect of AKT1 inhibition in vitro on tumor cell proliferation in vivo

Abstract Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with subtherapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multiscale, molecular snapshot of this “AKTlow” cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously upregulate a host of other proteins and metabolites posttranscriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication. Mol Cancer Ther; 15(1); 142–53. ©2015 AACR.

Supplementary Figure S2. KDM4A depletion alters protein synthesis and enhances mTOR inhibitor sensitivity.

Akti-1/2 microvesicles in vitro cross-cell line bioactivity assay

Effect of AKTi microvesicles on cell vitality and cell colony counts after exposure to stress conditions in vitro

Supplementary Figure S1. KDM4A depletion and catalytic mutant alter the distribution of translation initiation factors.

Effect of microvesicle treatment in vitro on tumor cell proliferation in vivo

Supplementary Figure S2. KDM4A depletion alters protein synthesis and enhances mTOR inhibitor sensitivity.

Changes in RNA Pol II pausing after AKTi treatment

Bioactivity and effect of microvesicles on growth rate using MK-2206 inhibitor in vitro

Immunofluorescence images for both HCT116 and MCF7 cells, treated either with DMSO or Akti-1/2. Cells were stained for DAPI, MCM2, H3K9me2, FDFT1 (A), and MCM2, H3K9me2, cholesterol, and lipid rafts (B)

The transcription factor NRF2 is a master regulator of cellular responses to oxidative stress, contributing to the pathogenesis of autoimmunity, metabolic disorders, and neurodegeneration. Somatic alterations in the NRF2 pathway also contribute to the growth and metastasis of many cancer types including ~30% of lung cancers. Still, the activation of NRF2 has frequently been observed in the absence of known genomic alterations, indicating that other pathways may drive its dysregulation. Further, approaches to target NRF2 pharmacologically have remained elusive. Here, we conducted a screen that identified a small molecule, ML329, exhibiting selective cytotoxicity in cells exhibiting NRF2 dependency and synthetic lethality to NRF2 pathway mutations across 489 cell lines. Surprisingly, we find that melanomas—which rarely have somatic mutations in the NRF2 pathway—were commonly sensitive to ML329. Melanomas were seen to exhibit NRF2-dependent metabolomic and transcriptional programs through the transcriptional activation of the adaptor protein p62/SQSTM1 by the melanocyte master regulator and oncoprotein MITF. This pathway was found to be conserved among all cancers characterized by genomic alterations of the MiT family (MITF, TFEB and TFE3) including subsets of renal cell carcinomas, pediatric sarcomas, and uveal and cutaneous melanomas. Our data identify a previously unrecognized, non-canonical mechanism of NRF2 activation by the MiT family, clarifying the regulation of NRF2 in pathologic and physiologic contexts. Pharmacologic inhibition of NRF2 could be valuable in the treatment of conditions with MITF family dysregulation. Citation Format: Xinbo Luo, Bart Lutterbach, Priya Pancholi, Yeon Sook Choi, Xiao Liu, Phillip Munson, Saqib Faisal, David A. Whipple, Robert A. Smith, Warren S. Weiner, David K. Johnson, Myriam Boukhali, Nicole S. Persky, Matthew G. Rees, Shunsuke Kitajima, David Barbie, Anuradha Roy, Michael Baltezor, Lian Rajewski, William McGuinness, John Haslam, Ananthan Sadagopan, Charles H. Yoon, Cory M. Johannessen, Christine G. Lian, Jason L. Hornick, Srinivas R. Viswanathan, David Liu, Vicki Nienaber, Wilhelm Haas, Frank J. Schoenen, David E. Fisher, Rizwan Haq. A non-canonical MiT/TFE-dependent NRF2 program is a druggable vulnerability in multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1706.

Summary of GSEA results for GRO-Seq, RNA-Seq and protein datasets

Supplementary figures and datasets legends

Lists of analytes (i.e., mRNA transcripts, proteins, metabolites, small RNAs) with concordant behavior

Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, is elevated in quiescent and chemo-treated leukemic cells and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. We find that FXR1 depletion reduces translation, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). FXR1 regulates factors that promote transcription and processing of ribosomal genes and snoRNAs. Ribosome changes in FXR1-overexpressing cells, including RPLP0/uL10 levels, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling selective translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these genes or phospho-eIF2α with inhibitors reduces chemosurvival. Thus, elevated FXR1 in quiescent or chemo-treated leukemic cells alters ribosomes that trigger stress signals to redirect translation for chemosurvival.