Carfilzomib Overcomes Resistance to Bortezomib in the Human Lagk-1A Multiple Myeloma Xenograft Model

Introduction: We previously demonstrated that when carfilzomib (CFZ) was administered first to SCID mice bearing our human multiple myeloma (MM) xenografts, followed by the addition of melphalan (MEL) at the time of disease progression, a modest further reduction in tumor size was observed compared with continuing CFZ or adding MEL alone. In addition, substitution of single agent bortezomib (BOR) for CFZ showed no anti-MM effects. However, when MEL was administered first and CFZ was added after disease progression, mice treated with the combination showed a reduction in tumor volume compared with mice that discontinued MEL. Mice treated with CFZ alone showed no reduction in tumor size. Throughout the study, there was a trend toward smaller tumors among mice in which MEL was followed by the addition of CFZ when compared to mice in which MEL was continued or discontinued and treated with single-agent CFZ or vehicle alone. In the current study, we evaluated the anti-MM effects of CFZ, MEL and BOR for severe combined immunodeficient (SCID) mice progressing from BOR+MEL treatment using our human MM xenograft model LAGk-1A.

Methods: Each SCID mouse was surgically implanted with a 20 – 40 mm³ LAGk-1A tumor piece into the left hind limb muscle. Seven days post-implantation mice were randomized into treatment groups based on human immunoglobulin (Ig) G levels. Carfilzomib stock solution (2 mg/ml) was diluted to 3 mg/kg using 5% dextrose and administered twice weekly on two consecutive days (Sundays and Mondays) via intravenous (i.v.) injection.Bortezomib stock solution (1 mg/ml) was diluted to 0.25 mg/kg using NaCl and administered twice weekly (Sundays and Tuesdays) via i.v. injection.MEL stock solution (3 mg/ml) was diluted to 1 mg/kg using PBS and administered once weekly (Fridays) via intraperitoneal injection. Mice (n = 10/group) were initially treated with the combination of BOR + MEL until tumor progression. Progression was defined as an increase in paraprotein > 25% confirmed on one consecutive assessment. After tumor progression, mice initially treated with BOR + MEL were randomized to continue to receive BOR + MEL, receive MEL only (discontinue BOR), receive BOR only (discontinue MEL), substitute BOR with CFZ, discontinue BOR + MEL and add in CFZ alone, or discontinue treatment altogether. Tumor size was measured using standard calipers and human IgG levels with an ELISA (Bethyl Laboratories, Montgomery, TX). This study was conducted according to protocols approved by the Institutional Animal Care and Use Committee.

Results: Following progression from the combination of BOR + MEL among mice bearing LAGk-1A, substitution of these drugs with single agent CFZ alone did not produce a reduction in tumor volume when compared with mice continuing to receive BOR + MEL. However, significantly smaller tumors were observed when mice progressing from BOR + MEL were switched to CFZ + MEL compared with mice continued on BOR alone (P = 0.0044) or animals switched to CFZ alone (P = 0.05). There was a trend throughout the study toward smaller tumors in mice receiving CFZ + MEL when compared to mice continuing on BOR + MEL, receiving BOR, CFZ or vehicle alone or discontinuing BOR + MEL. Following progression from BOR + MEL, similar anti-MM effects were observed on human IgG (paraprotein) levels among animals treated with CFZ + MEL when compared to mice continuing BOR + MEL, receiving BOR alone switching to CFZ alone or discontinuing both BOR and MEL.

Conclusions: These in vivo studies using our human MM xenograft model, LAGk-1A, show that SCID mice progressing from initial BOR + MEL treatment show a reduction in MM tumor burden when BOR is replaced with CFZ at the time of disease progression, and these tumors are significantly smaller than among mice continued on BOR + MEL. These studies demonstrate that once tumors progress from BOR + MEL treatment, CFZ can replace BOR and produce anti-tumor effects. A recent clinical trial confirms our preclinical findings and shows that most MM patients refractory to BOR in combination with other agents, including MEL, will respond to the substitution of CFZ for BOR (Berenson et al., Leukemia 2014).