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Integrated Phosphoproteomics and Transcriptional Classifiers Reveal Hidden RAS

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Integrated Phosphoproteomics and Transcriptional Classifiers Reveal Hidden RAS bioRxiv preprint doi: https://doi.org/10.1101/563312; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Lin et al. Page 1 Integrated Phosphoproteomics and Transcriptional Classifiers Reveal Hidden RAS Signaling Dynamics in Multiple Myeloma AUTHORS: Yu-Hsiu T. Lin1, Gregory P. Way2, Benjamin G. Barwick3, Margarette C. Mariano1, Makeba Marcoulis1, Ian D. Ferguson1, Christoph Driessen4, Lawrence H. Boise3, Casey S. Greene2,5, Arun P. Wiita1,* AFFILIATIONS: 1Department of Laboratory Medicine, University of California, San Francisco, CA 94107, USA 2Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA 3Department of Hematology and Medical Oncology and the Winship Cancer Institute, Emory University, Atlanta, GA, 30332, USA 4Experimental Oncology and Hematology, Dept. of Oncology and Hematology, Kantonsspital St. Gallen, St. Gallen, Switzerland 5Childhood Cancer Data Lab, Alex’s Lemonade Stand Foundation, Philadelphia, PA 19102, USA *CORRESPONDENCE: Arun P. Wiita, MD, PhD [email protected] UCSF Dept. of Laboratory Medicine 185 Berry St, Ste 290 San Francisco, CA 94107 1 bioRxiv preprint doi: https://doi.org/10.1101/563312; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Lin et al. Page 2 ABSTRACT: A major driver of multiple myeloma is thought to be aberrant signaling, yet no kinase inhibitors have proven successful in the clinic. Here, we employ an integrated, systems approach combining phosphoproteomic and transcriptome analysis to dissect cellular signaling in multiple myeloma to inform precision medicine strategies. Collectively, these predictive models identify vulnerable signaling signatures and highlight surprising differences in functional signaling patterns between NRAS and KRAS mutants invisible to the genomic landscape. Transcriptional analysis suggests that aberrant MAPK pathway activation is only present in a fraction of RAS-mutated vs. WT RAS patients. These high-MAPK patients, enriched for NRAS Q61 mutations, have inferior outcomes whereas RAS mutations overall carry no survival impact. We further develop an interactive software tool to relate pharmacologic and genetic kinase dependencies in myeloma. These results may lead to improved stratification of MM patients in clinical trials while also revealing unexplored modes of Ras biology. KEYWORDS: myeloma, phosphoproteomics, proteomics, kinase, RAS, KRAS, NRAS, MAPK, machine learning, signaling 2 bioRxiv preprint doi: https://doi.org/10.1101/563312; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Lin et al. Page 3 INTRODUCTION: Multiple myeloma (MM) is an incurable malignancy of plasma cells. Considerable effort has gone into deep sequencing of MM primary samples to identify genetic markers for classifying patients into groups for risk assessment and targeted therapy1-5. While these studies have offered significant insight into MM biology and prognosis, this knowledge from DNA sequencing largely remains untranslated into evidence-based therapeutic strategies. We hypothesized that one reason genomic profiles alone have not improved clinical outcome is that they may not be fully predictive of higher-level processes, such as dysregulated cellular signaling, that drive cancer phenotypes. For example, aberrant oncogenic signaling through protein phosphorylation can potentially be inferred, but not directly detected, at the genetic level. Mass spectrometry-based phosphoproteomics has therefore proven a powerful tool to explore cellular-wide signaling alterations at both baseline and under perturbation in cancer6,7. For example, studies in acute myeloid leukemia (AML) have shown that phosphorylation signatures can be used to predict increased sensitivity to kinase inhibitors both in cell lines and primary samples8,9. Alternatively, one indirect way to assess cellular signaling via a particular pathway is through gene expression profiling. While RNA expression levels of the genes encoding kinases themselves often do not correlate with pathway activation10, downstream transcriptional signatures of disparate genes induced by the signaling cascade may reveal specific functional readouts. In MM, it is thought that aberrant signaling is strongly driven by mutations in the RAS family of proto-oncogenes. These mutations are proposed to activate oncogenic signaling primarily via the RAS-REF-MEK-ERK (MAP kinase (MAPK)) pathway and the PI3K-AKT pathway11,12. Yet an outstanding mystery in oncology is tumor-specific selection of mutations in different RAS isoforms despite >80% sequence homology and 3 bioRxiv preprint doi: https://doi.org/10.1101/563312; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Lin et al. Page 4 highly similar biological function12,13. For example, epithelial-origin tumors predominantly harbor mutations in KRAS, whereas NRAS mutations are more common in hematologic malignancies, and HRAS mutations are rare overall12. Furthermore, it is not known why mutations in specific RAS codons are predominantly found in some cancer types (for example, KRAS G12D in pancreatic cancer, KRAS G12V in lung cancer, or NRAS Q61 in melanoma)14,15. MM is a unique case study of Ras biology as approximately 40% of MM patient tumors have predicted activating mutations in KRAS or NRAS4,5, with an almost equal distribution between the two, but essentially none in HRAS. Notably, these KRAS and NRAS mutations are very rare in the precursor lesion monoclonal gammopathy of uncertain significance (MGUS), suggesting that RAS mutations are important for transformation to MM16. The majority of MM research has treated mutations in these RAS isoforms as largely indistinguishable in terms of biological effects, though some studies have begun to elucidate distinctions. For example, clinical outcomes with earlier MM therapies showed patients with KRAS mutations had worse overall survival than those with NRAS mutations17,18. However, later clinical observations suggested that patients with NRAS mutations responded more poorly to bortezomib-based therapies than KRAS19. In vitro overexpression studies in myeloma cell lines indicated that KRAS may lead to more rapid proliferation in the absence of interleukin-6 (IL-6) stimulation than NRAS20,21. More recent sequencing studies have suggested that NRAS mutations tend to cluster with specific genomic aberrations in MM5. Finally, immunohistochemistry studies on MM bone marrow have suggested possible differences in ERK phosphorylation depending on RAS isoform and specific mutation22. Therefore, this evidence suggests that KRAS and NRAS in MM are not exactly equivalent, but much about the biology of these differences remains unclear. 4 bioRxiv preprint doi: https://doi.org/10.1101/563312; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Lin et al. Page 5 Here, we apply an integrated approach using both unbiased phosphoproteomics and machine learning-based classifiers of transcriptional response to dissect signaling in MM. Our results reveal differential kinase activity across MM cell lines with potential implications for selective kinase vulnerability. We next uncover underlying transcriptional output differences for patients with KRAS and NRAS mutations. Surprisingly, we find that only a fraction of RAS-mutated patients are predicted to have highly activated MAPK signaling vs. WT RAS patients. However, this group, which is particularly enriched in patients with NRAS Q61 mutations, carries the poorest prognosis under modern treatment strategies. Our results identify RAS-mutated MM patients who may benefit from precision medicine strategies and reveal modes of RAS isoform-driven biology with implications across RAS-mutated cancers. RESULTS: Kinase Activity from Phosphoproteomics is Modestly Predictive of Kinase Inhibitor Sensitivity in Multiple Myeloma Cell Lines Given promising prior results in AML8, we first aimed to predict differential kinase activity across MM cell lines and investigate whether increased kinase activity led to increased vulnerability to selective inhibitors. We used immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides across 7 MM cell lines (AMO-1, MM.1S, L363, INA-6, RPMI-8226, KMS-34, KMS-11) as well as one in vitro-evolved bortezomib-resistant cell line (AMO1br)23 (Fig. 1A). Phosphoproteomic profiling was performed in biological triplicate per cell line and in total 19,155 phosphosites from 4,941 proteins were quantified across all cell lines after imputation using MaxQuant24 and R25 software (Table S1). As expected with IMAC enrichment, >99% of all identified phosphorylation events were on Ser or Thr sites, with only a very small minority of Tyr sites. The Pearson R of phosphosite intensity correlations between replicates of each 5 bioRxiv preprint doi: https://doi.org/10.1101/563312; this version posted February
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