Kuiatse, Isere

Abstract Abstract 3723 Background: Waldenström macroglobulinemia (WM), a lymphoproliferative disorder with marrow infiltration by lymphoplasmacytic lymphoma cells and production of monoclonal IgM, is characterized clinically by good initial responses to standard therapeutic approaches. However, a minority of patients achieve a complete remission, and most inevitably relapse, indicating a need for validation of new, targeted therapies. Notably, B-cell receptor signaling has been linked to clonal evolution in WM, and Spleen tyrosine kinase (Syk) is over-expressed in primary patient cells. Also, a recent phase I/II study of the Syk inhibitor fostamatinib in patients with a variety of non-Hodgkin lymphomas showed evidence of activity in three patients with WM. These findings supported our central hypothesis, which proposed that Syk could be a rational new target for therapy of WM, which we pursued first with pre-clinical studies. Methods: Studies of fostamatinib were performed using the WM cell line MWCL-1, and the IgM-producing cell line BWCM.1, as well as in primary cells from patients with WM, and in a novel murine xenograft model utilizing MWCL-1 cells. Results: Fostamatinib induced a time- and dose-dependent reduction in viability of MWCL-1 and BWCM.1 cells, with median inhibitory concentrations (IC50) that were in the physiologically relevant range of approximately 0.25 and 1 μM, respectively, at 3 days. Cell cycle analysis showed a decrease in cells at S-phase and at G2/M, while an increase was seen in cells in G1, and in the sub-G1fraction, suggesting induction of apoptosis. Consistent with the possibility that there was activation of type I programmed cell death, increased staining was seen in fostamatinb-treated cells with an antibody to Annexin V. Interestingly, evidence was seen of the induction of type II programmed cell death, or autophagy, as measured by an increase in cells staining with the lysotropic dye acridine orange. At the molecular level, fostamatinib blocked signaling through p44/42 mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein kinase B/Akt, and Zeta-chain-associated protein kinase (ZAP)-70/Syk, as judged by a reduction in the phosphorylated forms of these intermediates by Western blotting. Notably, the viability of primary cells isolated from patients with WM was also reduced in association with blockade of p44/42 MAPK. Finally, a novel murine xenograft model was generated by injection of MWCL-1 cells into immunodeficient mice, which developed measurable tumors. When these were treated with fostamatinib, a delay in tumor growth was demonstrable. Conclusions: Targeting Syk with fostamatinib is an attractive strategy against pre-clinical models of WM both in vitro and in vivo. These findings lend further support for translation of fostamatinib to the clinic for patients with relapsed, and possibly even newly diagnosed WM. Disclosures: No relevant conflicts of interest to declare.

Abstract Purpose: Waldenström's macroglobulinemia is an incurable lymphoproliferative disorder driven by an L265P mutation in the myeloid differentiation primary response gene 88 (MYD88), which activates downstream NF-κB signaling through the Myddosome. As this pathway depends in part on activity of interleukin-1 receptor-associated kinases (IRAKs)-1 and -4, we sought to evaluate the potential of the IRAK1/4 inhibitor R191 in preclinical models. Experimental Design: Patient-derived cell lines and primary samples were used in both in vitro and in vivo experiments to model Waldenström's macroglobulinemia and its response to IRAK1/4 inhibitors. Results: R191 induced a dose- and time-dependent reduction in viability of BCWM.1 and MWCL-1 Waldenström's cell lines, and suppressed activation of IRAK1/4. This was associated with cell-cycle arrest at G0–G1, reduced levels of cyclin-dependent kinases 4 and 6, and induction of apoptosis in cell lines and primary patient samples. Further downstream, R191 exposure led to reduced activation of NF-κB, and of protein kinase B/Akt/mammalian target of rapamycin signaling, whereas expression of a constitutively active Akt mutant induced R191 resistance. Gene expression profiling and gene set enrichment analysis revealed a signature consistent with inhibition of c-Myc and activation of the endoplasmic reticulum stress response. In both subcutaneous and systemic murine models of Waldenström's, R191 showed antitumor activity. Finally, the activity of R191 was enhanced when it was combined with novel chemotherapeutics such as bortezomib, afuresertib, and ibrutinib. Conclusions: Taken together, these data support the translation of R191 as an approach to target IRAK1/4 to the clinic for patients with Waldenström's macroglobulinemia.

Abstract Purpose: Waldenström macroglobulinemia (WMG) is a lymphoproliferative disorder characterized by good initial responses to standard therapeutics, but only a minority of patients achieve complete remissions, and most inevitably relapse, indicating a need for novel agents. B-cell receptor signaling has been linked to clonal evolution in WMG, and Spleen tyrosine kinase (Syk) is overexpressed in primary cells, suggesting that it could be a novel and rational target. Experimental Design: We studied the impact of the Syk inhibitor fostamatinib on BCWM.1 and MWCL-1 WMG-derived cell lines both in vitro and in vivo, as well as on primary patient cells. Results: In WMG-derived cell lines, fostamatinib induced a time- and dose-dependent reduction in viability, associated with activation of apoptosis. At the molecular level, fostamatinib reduced activation of Syk and Bruton's tyrosine kinase, and also downstream signaling through MAPK kinase (MEK), p44/42 MAPK, and protein kinase B/Akt. As a single agent, fostamatinib induced tumor growth delay in an in vivo model of WMG, and reduced viability of primary WMG cells, along with inhibition of p44/42 MAPK signaling. Finally, fostamatinib in combination with other agents, including dexamethasone, bortezomib, and rituximab, showed enhanced activity. Conclusions: Taken together, these data support the translation of approaches targeting Syk with fostamatinib to the clinic for patients with relapsed and possibly even newly diagnosed WMG. Clin Cancer Res; 21(11); 2538–45. ©2015 AACR.