(P 450) INVESTIGATING THE THERAPEUTIC POTENTIAL OF SMALL MOLECULE INHIBITOR INOBRODIB AS A TREATMENT ALTERNATIVE FOR CIC-DUX4 SARCOMA

Objective: CIC-DUX4 Sarcoma (CDS) is a newly recognized subtype of undifferentiated round cell sarcoma driven by the fusion between cell cycle regulator and transcriptional repressor Capicua (CIC) and pioneer transcription factor DUX4. The CIC-DUX4 oncoprotein contains the N-terminal DNA binding domain of CIC and C-terminal transactivation domain of DUX4 which interacts with histone acetyltransferase complex P300/CBP to induce transcriptional activation. Previous studies have shown that iP300W- a selective P300/CBP histone acetyltransferase (HAT) domain inhibitor-induces cell cycle arrest and prevents growth of CDS xenografts. However, the clinical utility of iP300W remains a concern as bio-similar compounds such as A-485 have exhibited several dose-limiting toxicities in pre-clinical models. Here we investigated the therapeutic efficacy of clinically available alternative called CCS1477 (Inobrodib) which is a selective small molecule inhibitor of the P300/CBP bromodomain. Inobrodib is currently being evaluated in phase I/II clinical trials for prostate cancer and hematological malignancies with early results demonstrating a favorable therapeutic ratio.

Methods: We modeled CDS by generating subcutaneous xenograft tumors in immunodeficient CIEA NOG mice. In total, 20 CIEA NOG mice were transplanted with CDS tumor derived NCC-CDS-X1C1 cells resuspended in a media/Matrigel mixture. After palpable tumor masses were detected at the site of injection, the animals were treated with CCS1477 (or vehicle control) once daily by oral gavage for 21 consecutive days. The drug dose (20mg/kg) was selected based on bioavailability data and maximum tolerability observed in previous pre-clinical studies. Tumor growth was measured weekly using digital calipers and the animals were monitored closely for adverse effects. At 21 days, or pre-determined humane endpoint, the animals were euthanized and tumors were collected for additional studies including immunohistochemistry and western blot. In parallel, CellTiter-Glo assays were used to assess CDS tumor cell viability in vitro following treatment with CCS1477, Romidepsin (histone deacetylase inhibitor; HDAC) and BMS986158 (bromodomain inhibitor).

Results: The P300/CBP bromodomain inhibitor, CCS1477, did not prevent growth of CIC-DUX4 Sarcoma xenografts. The relative tumor volume at completion of the study was used as criterion of efficacy. The average relative tumor volume of the CCS1477 group was not significantly different than the average relative tumor volume of the control group at day 16 post treatment initiation. Moreover, in our hands, drug treatment was associated with the development of a severe lung infection/abscess which occurred in 4/10 mice (40%) compared to 0/10 in the control group. In vitro, treatment with CCS1477 resulted in only modest reduction in cell viability (~30% at 10nM). Unexpectedly, a control group treated with a selective class 1 HDAC inhibitor Romidepsin yielded the most significant reduction in CDS tumor cell viability. These results suggest that opposed to depleting acetylation with the use of P300/CBP inhibitors, hyperactivating the CDS transcriptional program by increasing histone acetylation could be a more effective therapeutic strategy in CDS.

Conclusion: P300/CBP has been identified as a potential therapeutic target for CDS. Previous data showed that small molecule inhibition of the of the P300/CBP HAT domain using iP300W induces cell cycle arrest and prevents growth of CIC-DUX4 Sarcoma xenografts. Conversely, our studies indicate that treatment with P300/CBP selective bromodomain inhibitor has no effect on tumor growth and is associated with high rates of toxicity in mice. Corollary in vitro studies suggest instead that HDAC inhibitors, which likely work by hyperactivating the CDS transcriptional program, could represent a more efficacious treatment alternative.