LAUSR

Recently, Hosen N et al. reported frequent expression of activated integrin β7 in multiple myeloma (MM) patients though the clinical significance was not known [1]. They are also conducting a clinical trial using CAR-T-cell therapy targeting activated integrin β7 [1, 2]. Here we report association of expression of integrin β5 and/or β7 with lenalidomide resistance. Lenalidomide has played a central role in MM therapy, but long-term use has also led to lenalidomide-resistance. Although several clinical studies have reported down-regulation or genetic mutation of cereblon (CRBN) as a mechanism of resistance, other molecular mechanisms have not been fully explored [3]. To address these questions, we established four MM cell lines by exposure to low-dose lenalidomide. As shown in Fig. 1a, a significant decrease in sensitivity to lenalidomide was observed in KMS21R, MUM24R and KMS27R compared with their parental cells. Both KMS34R cells and their parental KMS34 cells were resistant to even high-concentration lenalidomide. In addition, lenalidomide resistance is likely irreversible even after a month-long washout period (Fig. S1). Resistance to other anti-myeloma drugs was also examined. KMS21R, MUM24R and KMS34R cells tended to be more resistant to panobinostat than their parental cells (Fig. S2). MUM24R, KMS27R and KMS34R cells also became more resistant to bortezomib. Next, we examined expression of CRBN and the downstream molecules in these cells. As shown in Fig. 1b, expression of CRBN was decreased in KMS21R cells; conversely, expression of IKZF1, IKZF3, c-Myc and IRF4 was increased when compared with parental KMS21 cells. No significant change in expression of CRBN axis proteins was observed in MUM24R cells. The IKZF1 level increased and its degradation was delayed in KMS27R cells (Fig. 1b). To summarize, patterns of altered expression of CRBN and its downstream molecules after long-term culture with lenalidomide varied among the cell lines. To elucidate additional molecular mechanisms for lenalidomide resistance, we performed RNA sequence analysis using MUM24R and parental cells because it appeared that the CRBN axis was not responsible for drug resistance in these cells. Functional annotation clustering using the Database for Annotation, Visualization and Integrated Discovery (DAVID) showed higher expression of genes related to “cell adhesion” and “integrin-mediated signaling pathway” in MUM24R cells than in their parental cells (Fig. S3a). Since both pathways included integrin β5 and β7 genes, integrin signaling was thought to be a candidate for lenalidomide resistance. RNA sequence data revealed that expression of integrin β5 and β7 gene was up-regulated in MUM24R and KMS21R compared with their parental cells. In KMS27R and 34R, integrin β5 gene expression was increased despite lowered expression of integrin β7 gene (Fig.S3b). Functional annotation clustering also showed genes related to “cell adhesion” or related clusters and/or “integrin-mediated signaling pathway” in KMS21R, KMS27R and KMS34R cells (Fig.S3c). Expression of integrin β5 and β7 gene products were validated by western blot (Fig. 1c). Although RNA sequence data suggested increased expression of integrin β5 and β7 transcripts in MUM24R cells, expression of integrin β5 protein was not detected by western blot, and integrin β7 signals were very weakly detected only when MUM24 and MUM24R cells were exposed to lenalidomide (Fig. 1c). Since these integrins were significantly expressed in other lenalidomide-resistant cells (Fig. 1c), we continued further analyses. In order to elucidate whether expression of integrin β5 and β7 is associated with lenalidomide resistance, * Yutaka Hattori [email protected]