Systems Biology Analysis Identifies Targetable Vulnerability Networks to Proteasome Inhibitors in Multiple Myeloma
Authors: Meads Mark B. , Renatino Canevarolo Rafael , Sudalagunta Praneeth Reddy , Oliveira Paula S. , Magaletti Dario M. , Fang Bin , Coelho Siqueira Silva Maria D , Kulkarni Amit , Dai Hongyue , Dalton William S. , Petre Ion , Koomen John M. , Siqueira Silva Ariosto , Shain Kenneth H. 
Published: October 2019

Abstract
Proteasome inhibitors (PI) such as bortezomib and carfilzomib are critical components of anti-multiple myeloma (MM) therapy, yet all MM patients eventually develop refractory disease. We developed a non-biased method to identify and validate dysregulated pathways associated with PI-resistance in myeloma by combining RNAseq data from 522 MM patient specimens obtained from our Total Cancer Care/M2Gen/ORIEN network at Moffitt Cancer Center with paired ex vivo sensitivity to PIs and kinase inhibitors (KI). Dimensionality reduction analysis (t-SNE) and Fuzzy C-means was used to identify 422 clusters of genes that co-express in individual patients, and Gene Set Enrichment Analysis (GSEA) was used to identify clusters with gene expression patterns that correlated with PI sensitivity. Using publicly curated databases and in silico integrative analyses, we built protein-protein interaction networks to identify putative transcription factors, corresponding master regulators (kinases), and candidate KIs to promote PI sensitization. This systems biology approach identified a Chk1-Cdk1-Plk1 circuit associated with PI-resistance and also found 21 additional kinases (of 501 expressed in our cohort's kinome) that could be targeted to re-sensitize PI-resistant MM, which we confirmed in cell lines, specimens from relapsed patients, and two in vivo models.
A panel of paired isogenic PI-resistant and sensitive MM cell lines were differentially screened to find kinases associated with PI-resistance using activity-based protein profiling (ABPP) and KI activity measured by high-throughput viability assay. The MM cell lines 8226 and U266, along with their drug resistant counterparts 8226-B25 and U266-PR, were grown in mono-culture for 24h and lysates were enriched for ATP binding proteins by affinity purification versus a chemical probe. Tryptic peptides were measured using discovery proteomics (nano-UPLC and QExactive Plus mass spectrometer) to identify 85 kinases out of a total of 715 proteins in 8226-B25 MM cells and 35 kinases out of a total of 688 proteins in U266-PR MM cells that were preferentially enriched by 2-fold change compared to parental cell lines. Twenty-four kinases were commonly activated among PI-resistant cell line pairs and were screened in PI-resistant myeloma lines using a label-free, high throughput viability assay that simulates the tumor microenvironment. Three KIs targeting Plk1 (volasertib and GSK461364) and Cdk1/5 (dinaciclib) consistently maintained LD50s in the low-nanomolar range and induced caspase-3 activation in four PI-resistant MM cell lines: 8226-B25, U266-PR, ANBL-6-V10R, and Kas6-V10R.
Twenty-four kinases each were identified by RNAseq/ex vivo PI sensitivity of MM specimens and ABPP of PI-resistant/sensitive MM cell line pairs. Of these, 7 kinases were identified by both methods: Cdk1, Chk1, Plk1, ILK, Syk, PKA, and p70S6K. Several KIs targeting Cdk1, Plk1, ILK, DNAPK, Syk, MKK7, Nek2, and mTOR identified in patient specimen or cell-line screens showed single agent activity in MM patient bone marrow specimens purified by a CD138 affinity column. Among these, inhibitors to Cdk1, ILK, mTOR, and Plk1 showed the most activity in patient specimens with an average 96h LD50 of 25 nM (n=56), 2.4 uM (n=42), 2.7 uM (n=57) and 3.8 uM (n=53), respectively. Six KIs targeting Plk1, ILK, Syk, MKK7, Nek2 and MARK3 were synergistic with carfilzomib in 20 patient specimens and maintained or improved ex vivo activity in relapsed refractory MM (RRMM) specimens. Volasertib, which targets Plk1, was the most synergistic with carfilzomib of all KIs tested in patient specimens and was further validated in two in vivo models: a NSG/U266 xenograft model of PI resistance and the syngeneic C57BL/6-KaLwRij/5TGM1 immunocompetent model. Volasertib significantly increased survival and reduced tumor burden in both models as a single agent, and was more effective versus PI-resistant tumors compared to PI-sensitive counterparts. Our pharmaco-proteomic screen, coupled with rich gene expression data from patients identified Plk1 as a target critical to MM survival in the context of acquired PI resistance and represents a unique workflow to find tumor vulnerabilities that arise during therapy. We anticipate that these data will also produce a critical path for the personalized allocation of therapy to maximize efficacy and minimize the use of ineffective therapies in RRMM.

Disclosures
No relevant conflicts of interest to declare.