Founding Precision Therapy in 1q-Amplified Multiple Myeloma

Introduction

Multiple myeloma is an incurable plasma cell malignancy with a strikingly heterogeneous genomic landscape. Other than IgH translocations and hyperdiploidy, only a few alterations are observed in large enough numbers. Amplification of the long arm of chromosome 1 (1q) is among the most common copy number alterations encountered, with a confirmed adverse effect on survival. Gene expression profiling has identified a minimal common amplified region between 1q21 and 1q23 as a probable target of the amplification event, however the actionable gene dependencies in that region have not been explored. In this study, we employ a large number of in-house and publicly available CRISPR, shRNA and drug screens in an effort to characterize the genetic dependencies of 1q-amplified myeloma and discover drugs that target them. Ultimately, we hope to propose a tailored therapeutic strategy for patients with 1q-amplified multiple myeloma.

Methods

To assess the genetic dependencies of 1q-amplified myeloma, we performed an shRNA screen in multiple myeloma cell lines, targeting genes in the 1q21-1q23 region. Corresponding C911 hairpins were designed for every target shRNA, and DEMETER2 was used to infer on-target effect. To that same end, we analyzed publicly available dependency data from Project Achilles (Whole-genome CRISPR screen, Avana library, 18Q4 release) and Dependency Map (combined RNAi dataset, accessed on 6/20/2018) and looked for differential dependencies in 1q-amplified multiple myeloma cell lines. Different sets of 1q-amplified and non-amplified cell lines were included in each dataset to avoid cell line-specific effects. Genes that both constituted differential dependencies and were differentially expressed were considered as hits. GSEA was used for pathway analysis.

To assess differential sensitivity of 1q-amplified myeloma to drugs, we performed a drug screen utilizing the Broad Institute's Drug Repurposing Library-a library of over 5,000 drugs that have cleared varied stages of clinical testing, and compared normalized viability values between 1q-amplified and non-amplified myeloma cell lines. Utilizing publicly available patient data, we also built a 1q-amplification gene expression signature and used it to query the Connectivity Map (CMap) database. Drugs that were predicted to reverse our signature were then used in a new drug screen of myeloma cell lines.

Results

Through multiple dependency screens, we identified a total of 206 differential dependencies in 1q-amplified myeloma. Out of those, 46 came up in two screens (double hits), while 4 came up in all three datasets (triple hits). CLK-2, a serine/threonine and tyrosine kinase involved in mRNA splicing and POLR3C, a gene encoding a subunit of RNA-polymerase III, were among the triple hits. MCL-1, UBQLN4, CERS2, JTB, BCL9 and PEX19 were among the double hits. With at least four members affected (UBQLN4, UBE2Q1, UBAP2L and UBE2T), the ubiquitin pathway came up as an important differential dependency, while GSEA identified cell cycle as another pathway of essentiality in 1q-amplified multiple myeloma.

Next, we searched for differential drug sensitivities utilizing the Drug Repurposing Library as well as a CMap-guided screen, as described above. We identified as hits several compounds targeting the MDM2 ubiquitin ligase as well as compounds related to cell cycle control, including PARP inhibitors and chemotherapeutic agents like fludarabine, thus validating the dependencies discovered in our datasets.

Conclusion

We employed a combination of multiple in-house and publicly available CRISPR, shRNA and drug screens, in the largest to date effort to characterize and target the genetic dependencies of 1q-amplified multiple myeloma. Cell cycle and the ubiquitin pathway came up as strong dependencies, while the drugs that target them were indeed shown to preferentially kill 1q-amplified myeloma cell lines. Thus, for the first time, our results suggest that patients with 1q-amplified myeloma might benefit from genetically tailored treatment involving cell cycle and ubiquitin inhibitors or a combination thereof. And inasmuch as 1q amplification is one of myeloma's few frequent alterations, this discovery has the exciting potential to affect change in a large number of patients.