Hypoxia Activated Prodrug TH-302 for the Treatment of Multiple Myeloma.

Abstract 3838

Poster Board III-774

Hypoxia is known to be linked to increased metastatic potential and a treatment-resistant phenotype leading to rapid progression and poor prognosis in solid tumors. We confirmed previous data[1] on hypoxia in human multiple myeloma (MM) in the 5T33MM syngeneic murine model of MM by using H&E staining and Hypoxyprobe (Pimonidazole) staining on consecutive serial sections from both naive mice and 5T33MMvv diseased mice. We observed a physiological hypoxic situation in MM diseased bone marrow. Given the contribution of hypoxia to tumor progression and drug resistance, a number of hypoxia-targeted therapeutics are under development. TH-302 is a new hypoxia-activated prodrug (HAP) that is currently being evaluated in the clinical trials as monotherapy and in combination with standard chemotherapy regimens for the treatment of solid tumors. The aims of the current study are (1) to demonstrate the effects of TH302 on MM cells in hypoxic conditions, focusing on apoptosis and cell cycle and associated signaling pathways and (2) to evaluate potential therapeutic effects when used in an experimental mouse MM model. We evaluated the effects of TH-302 in vitro on the murine 5T33MMvt cell line and the human LP-1, MMS-1, RPMI-8226, Karpas MM cell lines. Flow cytometry analysis revealed that TH-302 (0.5-50μM) can induce significant Go/G1 cell cycle phase arrest and apoptosis in hypoxic conditions (both 1% and 0% O2) in a concentration dependent manner, in contrast to normoxic conditions (20% O2) (p<0.001). Western blot confirmed that treatment with TH-302 in hypoxic conditions down-regulates cyclin D1/2/3, CDK4/6 and pRb expressions, but CDK2 expression was not disturbed. Furthermore, treatment with TH-302 in hypoxic conditions down-regulates the anti-apoptotic proteins BCL-2 and BCL-xL, as well as up-regulates the expression of three proapoptotic proteins: cleaved caspase-3, 9 and PARP. The expression pattern of Bax was however not influenced. The expression of p21 and p27 decreased in hypoxic condition after treatment with TH-302. Further studies conducted in the 5T33MMvv mouse model demonstrated that animals treated prophylactically with TH-302 (12.5 mg/kg, 25 mg/kg and 50 mg/kg, i.p.) for 3 weeks from day 1 after tumor inoculation showed decreased serum paraprotein (12.5 mg/kg, 32% decrease, p<0.05; 25 mg/kg, 77% decrease, p<0.001; 50 mg/kg, 54% decrease, p<0.001), compared to vehicle-treated 5T33MMvv mice (n=10). The frequency of apoptotic multiple myeloma cells in bone marrow sections was also significantly increased (12.5 mg/kg, 2.5 fold, p<0.05; 25mg/kg, 2.1 fold, p<0.05; 50mg/kg, 3.1 fold, p<0.01). Treatment with TH-302 resulted in no adverse events, any observable detriment to the mice or weight loss (p>0.05). In conclusion, these results show that hypoxia-activated treatment with TH-302 activates apoptosis and induces cell cycle arrest in MM cells, under hypoxic conditions, both in vitro and in vivo and therefore represents a promising therapeutic approach for multiple myeloma. Reference [1] Simona Colla, Paola Storti, Gaetano Donofrio, et al. Hypoxia and Hypoxia Inducible Factor (HIF)-1α in Multiple Myeloma: Effect on the Pro-Angiogenic Signature of Myeloma Cells and the Bone Marrow Microenvironment, 50th ASH annual meeting, http://ash.confex.com/ash/2008/webprogram/Paper13156.html