CRISPR-Cas9 Mediated CD45 Knockout Inactivates Src Family Kinases and Impairs Cell Migration in Multiple Myeloma

Abstract

IntroductionMultiple myeloma (MM) is a plasma cell malignancy that manifests continuous cell dissemination to multiple bone marrow (BM) niches and extramedullary (EM) sites. However, the molecular mechanisms behind this phenomenon remain elusive. CD45, a receptor tyrosine phosphatase, is an important regulator for T-cell and B-cell signaling pathways. In MM, the loss of CD45 expression has been correlated with earlier disease progression and inferior treatment outcomes. The downstream targets of CD45, Src family kinases (SFK), are associated with cell migration in many malignancies. It is also known to interact with proline-rich kinase (Pyk2) that binds to cytoskeleton-regulating proteins. Our previous in vitro studies demonstrated a ‘metastatic’ phenotype for CD45 negative MM. We hypothesise that CD45 acts as a marker for disease progression and mediates cell mobility through SFKs.MethodCD45 expression in human myeloma cell lines (HMCL) was assessed by flow cytometry. To investigate the functions of CD45 in MM, CRISPR-Cas9 mediated CD45 knockout (CD45KO) models were established from a HMCL, OCI-MY1. The resulting phenotypic and transcriptional changes were identified by immunoblotting, modified Boyden chamber assays, RNA sequencing and Phospho Explorer Antibody Array. SFK inhibitor (Saracatinib), Pyk2 inhibitor (PF573228) and siRNAs were used to validate the role of SFK (predominately Lyn and Fyn) and Pyk2 in migration.ResultsWe first compared 2 pairs of HMCL contemporaneously derived from BM and extramedullary disease (EM) in the same patient: TK1 (BM) and TK2 (EM), and TK17 (EM) and TK18 (BM). Both TK1 and TK18 had higher CD45 expression than their paired HMCL as expected.To avoid confounding intercellular genetic heterogeneity, we generated CD45KO models from OCI-MY1 and the loss of extracellular and intracellular portion of CD45 was confirmed by flow cytometry and immunoblotting. OCI-MY1 CD45KO cells showed significant SFK (Lyn and Fyn) and Pyk2 inactivation as compared to CD45 wild-type (CD45WT) cells. These cells demonstrated a significant reduction in homing capacity towards healthy and MM-patient derived BM stromal cells (reduced to 11.5% and 2.7%, p<0.0001, respectively) compared with the CD45WT cells. Treatment of CD45WT cells with Saracatinib and PF573228 similarly inhibited the homing capacity (47.3%, p<0.0001 and 71.3%, p<0.01 respectively). Moreover, silencing of Lyn and Fyn with siRNAs in CD45WT cells recapitulated the findings seen with the CD45KO cells (76%, p<0.01 and 55%, p<0.001, reduction in homing, respectively).The inactivation of SFK prompted us to investigate the transcriptional changes in the CD45KO cells. RNA sequencing identified differentially expressed migration-related genes in the CD45KO cell, in particular ADAM19, PARVB, AFAP1L2 and ITGAL, thus raising the possibility that dysregulation of these genes led to the observed reduction of homing potential of the CD45KO cells. To further understand the role of CD45 phosphatase activity, we analysed protein phosphorylation profiles in the CD45KO and CD45WT cells. In addition to the recognised inactivation of SFK and Pyk2, the Phospho Explorer Antibody Array also demonstrated reduced phosphorylation of JAK1 (Y1022), TYK2 (Y1054), c-Raf (S259) and MDM2 (S166), and increased phosphorylation of IRS-1 (S794), SHP-2 (Y580), c-Jun (Y170) and STAT6 (T645). We are currently investigating the correlation of these phosphorylation and MM homing mechanisms.ConclusionOur data demonstrate that CD45 plays an important role in regulating MM homing towards BM stroma by modulating SFK and Pyk2 activity with the loss of CD45 expression also resulting in the dysregulation of migration-related genes and widespread phosphorylation changes. Further in vivo studies evaluating the ‘metastatic’ impact of loss of CD45 expression are in progress. DisclosuresNo relevant conflicts of interest to declare.