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S1. Combination of lenalidomide (1uM) and bortezomib (different concentrations) induces apoptosis in myeloma cell lines (MM.1S and U266) compared to bortezomib alone treated cells (n=3) at the end of 48 hours. S2. Representative Immunoblot showing induction of apoptosis in MM.1S cell line upon treatment with lenalidomide (1uM) and bortezomib (5nM), lysates were collected the end of 24 and 48 hours post treatment with drugs (n=3). The proteins analysed were mentioned in the image. S3. a) Immunoblot showing that the combination of lenalidomide and bortezomib degraded IKZF1 in U266 cell line (n=3). b) Immunofluorescence assays showing degradation of IKZF1 in U266 cell line upon treatment with Lenalidomide and bortezomib (n=3). S4. Immunoprecipitation of Ubiquitin after treatment with lenalidomide (1uM) and bortezomib (5nM) revealed an interaction with IKZF1 at 8 hours. S5. Inhibition of autophagy by hydroxycholoroquine (HCQ) and bafilomycin A1 (BAFA1) in MM1.S cell line was confirmed using cyto-ID stain, where accumulation of autophagosome was observed (n=3) which was further confirmed by western blot (n=3). S6. Inhibition of autophagy by hydroxycholoroquine (HCQ) and bafilomycin A1 (BAFA1) did not inhibit the degradation of IKZF1 in U266 cell line (n=3). S7. Inhibition of calpain and caspase by PD150606 and zVAD.fmk inhibited the degradation of IKZF1 in U266 cell line (n=3). S8. Inhibition of calpain and chelation of calcium resulted in inhibition of PARP cleavage and IKZF3 degradation in MM1.S cell line (n=3) at the end of 24 hours of drug treatments. S9. Immunoblot representing modulation of calcium flux with ionomycin in MM.1S cell line enhanced IKZF1 degradation only in Ionomycin alone or in combination with LEN treated cells for 12 hours (n=3). S10. Viability of MM1.S cell line at post LEN (1uM) and BTZ (5nM) treatment after 48 hours (n=3). Viability was assessed by Annexin V- and 7 AAD - viability assay.

Multiple myeloma had been successfully treated by combining lenalidomide and bortezomib with reports suggesting benefits of such a combination even in relapsed/refractory cases. Recently, it was demonstrated that Ikaros degradation by lenalidomide happens via proteasome-dependent pathway and this process is critical for the eradication of myeloma cells. On the basis of this, an antagonistic effect should be observed if a combination of both these agents were used, which however is not the observation seen in the clinical setting. Our study demonstrates that when these agents are combined they exhibit a synergistic activity against myeloma cells and degradation of Ikaros happens by a proteasome-independent calcium-induced calpain pathway. Our study identifies the crucial role of calcium-induced calpain pathway in inducing apoptosis of myeloma cells when this combination or lenalidomide and bortezomib is used. We also report that this combination enhanced the expression of CD38 compared with lenalidomide alone. Thus, data from our study would establish the rationale for the addition of daratumumab along with this combination to further enhance therapeutic activity against multiple myeloma. Implications: Lenalidomide and bortezomib combination degrades IKZF1 in multiple myeloma through a calcium-dependent calpain and caspase pathway. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/18/4/529/F1.large.jpg.