A Novel Sequestosome-1/p62 ZZ Domain Inhibitor Induces New Bone Formation In The Presence Of Myeloma In Vivo

Multiple myeloma (MM) cells activate multiple signaling pathways in bone marrow stromal cells (BMSC), which results in enhanced tumor growth, increased osteoclast (OCL) formation and activity, and profound inhibition of bone formation by osteoblasts (OBL). We recently showed that sequestosome-1 (p62) acts as a signaling hub for these pathways, including MM-cell induced NFκβ, p38MAPK, and PI3K activation in MM patient BMSC, and that the p62 ZZ domain is required for BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. To target the ZZ domain we developed a small molecule inhibitor (CMPD3) that specifically blocked p62-ZZ domain mediated protein interactions required for BMSC support of tumor / OCL formation, but had no effect on CFU-blasts. We now report characterization of CMPD3 in an in vivomodel of MM.

Murine MM cells (5TGM1-tk-gfp) were implanted intra-tibially in female SCID mice and male C57BL/KaLwRij mice and allowed to engraft for 2 weeks. Pilot PK studies using a multiple dose MTD of 60mg/kg IV in female SCID mice bearing MM cells demonstrated a terminal t_1/2 of CMPD3 in plasma of greater than 5 hours (h), and extratibial tumor CMPD3 concentrations ranging between 5 and 10-fold higher than plasma. A pilot efficacy study in male C57BL/KaLwRij mice based on the multiple dose MTD demonstrated that mice treated with CMPD3 lost weight compared with vehicle treated mice. CMPD3 treatment did not alter activity scores of the mice or plasma IgG_2b levels, a marker of tumor burden. Both compartmental and non-compartmental pharmacokinetic analysis of plasma concentrations of CMPD3 in the male C57BL/KaLwRij mice on the multiple dosing regime after doses of 27 mg/kg/day or 40 mg/kg/day ip resulted in a plasma terminal t_1/2 of 10 -12 h and a clearance of 1188 ml/h/kg. An expanded efficacy study using CMPD3 doses of 27mg/kg/day or 40mg/kg/day, or vehicle ip for 5 days/week for 2 weeks demonstrated no change in body weights, tumor scores, or plasma IgG_2b levels in any animals.

Radiographic analysis of tibiae confirmed the presence of lytic bone lesions in all animals, and of 27 evaluable animals at the end of treatment, 6 CMPD3 treated and 2 vehicle treated mice developed grade 4 tumors (defined as tumor expanding outside the tibia and impairing the use of the limb). 4 of the 6 CMPD3 treated mice with grade 4 tumors and 1 of the 2 vehicle treated animals had radiographic evidence of new bone formation on the anterior tibial surface. µCT analysis of the tibiae from all CMPD3 vs. vehicle treated mice showed that the ratio of new bone volume to total bone volume (NBV/TBV) was increased in CMPD3 treated mice regardless of tumor score (1 - 4) (p=0.015). Stratification of NBV/TBV based on tumor score showed that CMPD3 treated animals with a tumor score of 4 had significantly more new bone formation than animals with tumor scores of 1 - 3 (p<0.01). NBV/TBV was not increased in the non-injected tibiae, indicating a lack of systemic effect on non tumor-bearing bone.

Thus, CMPD3 induces new bone formation in bones bearing MM and suggest that p62 ZZ inhibition modulates tumor induced OBL suppression. Non-compartmental analysis of the maximal plasma concentration of CMPD3 at the 40mg/kg dose was 5.7µM, while the in vitro IC₅₀ of CMPD3 for 5TGM1 cells was 4.35µM. This difference may explain why IgG_2b levels did not change with CMPD3 treatment. In contrast, the IC₅₀ in OCL formation assays was 0.1µM, suggesting that CMPD3’s inhibition of OCL formation in vivo may also contribute to increased bone. These findings demonstrate that CMPD3 can modulate tumor cell – bone cell interactions in MM in vivo, and support development of CMPD3 as a potential anabolic bone agent for MM.