S135: CRISPR/CAS9 EDITING REVEALS DEPENDENCE OF HUMAN RICHTER SYNDROME AND MURINE CLL CELLS ON SIGNALS FROM B CELL RECEPTOR, CXCR4 RECEPTOR AND MACROPHAGES BUT NOT FROM TOLL-LIKE RECEPTORS IN VIVO

Abstract

Background: Numerous signals from the microenvironment have been identified that can increase the survival or induce the proliferation of chronic lymphocytic leukemia (CLL) cells in vitro. These signals typically represent various secreted or cell surface ligands that are expressed by different cell types present in the lymph node tumor microenvironment, such as T cells, macrophages and stromal cells, or molecules that would be expected to be released by apoptotic cells, such as apoptosis associated autoantigens or CpG-unmethylated mitochondrial DNA. However, the extent to which these different signals contribute to the growth and survival of the leukemic cells in vivo has still not been fully established. Aims: To determine the relevance of signals from Toll-like receptors (TLRs), the B cell receptor (BCR) and the chemokine receptor CXCR4 for the growth of murine Eμ-TCL1 CLL cells and human Richter Syndrome (RS) patient-derived xenograft (PDX) cells in vivo. Methods: To understand the impact of pharmacological inhibition of the TLR pathway, immunocompetent C57BL/6 or immunodeficient NSG mice were transplanted with murine Eμ-TCL1 CLL or human RS-PDX cells, respectively, and were treated with an inhibitor of the kinase IRAK4 or vehicle control. To determine the effects of genetic disruption of the TLR, BCR and CXCR4 pathways, the IRAK4, IgM heavy chain (IGHM) and CXCR4 genes were disrupted by CRISPR/Cas9 editing in the murine Eμ-TCL1 CLL cells or the human RS-PDX lines RS9737, RS1316 and IP867/17 prior to transplantation. The effects of genetic disruption of these pathways on the growth of the malignant cells in vivo were investigated by analyzing the proportion of mutant and wild type alleles in different anatomical compartments of the transplanted mice. Results: Treatment with the IRAK4 inhibitor significantly prolonged the survival of C57BL/6 mice transplanted with murine Eμ-TCL1 CLL cells and significantly delayed the growth of the human RS-PDX lines RS9737 and RS1316 xenografted in immunodeficient NSG mice. However, genetic disruption of IRAK4 in the human RS-PDX lines RS9737, RS1316 and IP867/17 or of the TLR adaptor MyD88 in the murine Eμ-TCL1 CLL cells did not result in negative selection of these cells in vivo, suggesting that these tumors do not receive or do not rely on TLR signals for their growth and that the therapeutic activity of the IRAK4 inhibitor is not caused by disruption of TLR signaling in the malignant cells themselves. In contrast, genetic disruption of the IGHM or CXCR4 gene was associated with significantly reduced growth of these cells compared to their wild type counterparts and resulted in almost complete disappearance of the mutated cells at later timepoints following transplantation. Analysis of the effects of the IRAK4 inhibitor on other cell types from the tumor microenvironment revealed a significant reduction in the number of macrophages, which in co-culture experiments were shown to strongly protect human Richter syndrome and murine Eμ-TCL1 leukemia cells from spontaneous apoptosis. Summary/Conclusion: These data provide evidence that signals from the BCR, CXCR4 and macrophages support the growth and survival of CLL and RS cells in vivo and argue against a role for TLR signals in the pathogenesis of CLL. In addition, they suggest that targeting the TLR pathway may potentially provide a therapeutic benefit in CLL, but that this benefit would be derived from inhibition of TLR signaling in monocytes and macrophages rather than inhibition of TLR signaling in the malignant cells themselves.