7025 Background: BTX-1188 is a first-in-class oral molecular glue that degrades GSPT1 and IKZF1/3 and is currently in phase 1 clinical trials for treatment of hematologic and solid malignancies. Targeted protein degradation of cereblon neosubstrates is clinically validated in the treatment (tx) of hematologic malignancies (Lu 2014, Zou 2020). Methods: Cell viability in BTX-1188-treated cells and patient samples was measured by CellTiter-Glo 2.0 assay (Promega). Substrate degradation and apoptosis profiles were analyzed by immunoblots of protein lysates from cells treated with DMSO or BTX-1188. Vehicle, or 30 or 40 mg/kg IP BTX-1188, was used in athymic nude mice AML xenograft models. Results: BTX-1188 is a rapid, deep, and potent degrader of GSPT1 and IKZF1/3 and inhibitor of Myc in several cancer cell lines (Table). Proteomics and immunoblot analysis of AML cell line, MV-4-11, shows significant degradation of GSPT1 and IKZF1 after 2 h tx with 100 nM BTX-1188 ( P<1x10-5) and 6 h tx with 3 nM BTX-1188 (>90% of GSPT1), respectively, indicating rapid and potent neosubstrate degradation. BTX-1188 also durably degrades GSPT1 where tx with 30 nM for 6 h followed by washout maintains significantly lower levels of GSPT1 and sustained apoptosis for up to 24 h. Owing to IKZF1/3 degradation, BTX-1188 has immunomodulatory properties as seen by inhibition of proinflammatory cytokines (IL-1β, IL-6, TNFα) and induction of IL-2 by LPS and αCD3-stimulated PBMCs, respectively. This approach is expected to improve clinical outcomes and reduce toxicities associated with pure GSPT1 degradation (CC-90009), thus expanding the therapeutic window of BTX-1188. Functionally, BTX-1188 is cytotoxic in various cancer cell lines such as Myc-driven lines (IC50 range: 0.5-10 nM) and primary human AML patient samples (IC50 range: 0.4-1.5 nM), including relapsed/refractory-, cytarabine- and venetoclax-resistant samples. The durability of GSPT1 degradation and sustained apoptosis in response to BTX-1188 tx is further reflected in in vivo efficacy models where daily or intermittent dosing of BTX-1188 results in potent and sustained antitumor activity. Conclusions: These preclinical data show that BTX-1188 is a promising drug candidate for AML and other tumor types. Its immunomodulatory properties owing to IKZF1/3 degradation may prevent systemic inflammatory dose-limiting toxicities associated with pure GSPT1 degradation (Uy 2019). BTX-1188 has entered phase 1 clinical studies for advanced solid tumors and AML. DC50 (nM) or inhibitory concentration (nM) of BTX-1188 at 6 h of Tx. [Table: see text]
BioTheryX's novel and unique Protein Homeostatic Modulators (PHMsTM) are small molecule “molecular glues” that promote ubiquitination of substrate proteins by the CRL4CRBN (CUL4-DDB1-RBX1-CRBN) ubiquitin ligase, leading to their subsequent degradation by the 26S proteasome. The therapeutic benefit of targeted protein degradation via cereblon (CRBN) has been clinically validated by the immunomodulatory drugs lenalidomide and pomalidomide in 5q-deletion-associated myelodysplastic syndrome and multiple myeloma, respectively. We present PHM® therapeutic candidates with novel targeted degradation profiles of disease-relevant proteins including GSPT1, Ikaros, and CK1α via CRBN with significant clinical potential in the treatment of hematological malignancies as well as solid tumors. Phenotypic screens of our proprietary PHM® library has led to the discovery of compounds that exhibit significant cytotoxicity in human AML and lymphoma cell lines, unlike classical IMiDs such as lenalidomide and pomalidomide. Cell viability assays revealed that these PHMsTM have IC50s in the low-nanomolar range. Importantly, they exhibit a large in vitro safety window with higher IC50s in normal human liver epithelial cells. The compounds are highly potent and can achieve significant AML and lymphoma selective cytotoxicity with short-term exposure of only 8 hours suggesting a highly rapid mechanism of action. Immunoblot analysis shows that the PHMsTM degrade CRBN substrates such as GSPT1, Ikaros and CK-1α and concomitantly activate the apoptosis machinery within 6 hours of treatment. The substrate degradation profile of PHMsTM combined with the cytokine profile divides the PHMsTM into two distinct mechanistic and functional classes of molecules - (1) purely cytotoxic, and (2) cytotoxic and immune-modulatory PHMsTM. Human PBMCs activated with lipopolysaccharide or α-CD3 followed by DMSO or compound treatment for 24 hours revealed that PHMsTM belonging to the latter class inhibit proinflammatory cytokines such as IL-1β, IL-6 and TNF-α as well as induce IL-2, an indicator of T cell activation, and are a 100-fold more potent than pomalidomide. In vivo efficacy of BTX-PHMsTM was evaluated using the MV-4-11 human AML xenograft model in athymic nude mice. The study shows a significant reduction in tumor volume with daily dosing, further establishing the PHMsTM as clinical candidates for AML. Of significance, is the effect of BTX1188 that completely obliterates the tumors within 30 days of dosing and maintains tumor free animals until the end of the study. The effective short-term exposure in vitro is also reflected in vivo where intermittent dosing with the PHM results in significant reduction in tumor volume. Finally, BTX-PHMsTM exhibit significant oral bioavailability, thus making them promising clinical candidates for treatment of hematological malignancies as well as early discovery candidates for solid tumors. Citation Format: Aparajita Hoskote Chourasia, Leah Fung, Angela Pasis, Brooke McElwee, Angela Schoolmeesters, Normand Richard, Imelda Lam, Eduardo Torres, Paul Erdman, Robert Sullivan, David Hecht, Kyle W. Chan, Frank Mercurio, David I. Stirling. Targeting hematological malignancies with two functionally and mechanistically distinct classes of cereblon mediated protein homeostatic modulators [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1963.
CDK4/6 inhibitors (CDK4/6i) such as palbociclib and ribociclib are used to treatER+/HER2- breast cancer, but patients can develop resistance via mechanismsincluding the INK4-CDK6 complex which have been shown to limit the effectiveness ofCDK4/6i in ER+ breast cancer. Up to 20% patients exhibit innate resistance and up to70% patients develop acquired resistance after 3 years on therapy (Scheidemann,2021). To address this limitation, we utilized our PRODEGY platform of Cereblon(CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders topotently inhibit tumor growth in CDK4/6i resistant tumors and in treatment naïveER+/HER2- breast cancer and triple negative breast cancer (TNBC). We examined theeffect of our degraders on multiple nodes of this pathway. Target degradation byimmunoblot analysis of TNBC cell line, MDA-MB-231, treated with our CDK4/6bifunctional degraders for 6 hours showed over 50% degradation of CDK4 and CDK6at 10-100nM. Active CDK4/6 phosphorylates the protein RB which releases thetranscription factor E2F, inducing the expression of genes which promote cell cycleprogression. Analysis of RB phosphorylation by in-cell western upon 24 hour ofCDK4/6 degrader treatment showed phospho-RB IC50s at <50nM. Cell cycle analysisby staining with propidium iodide after 24 hours of treatment with CDK4/6 degradersinduced G0/G1 cell cycle arrest at concentrations as low as 10nM. As a readout forgrowth inhibition through cell cycle arrest, we examined the ability of these degradersto inhibit the proliferation of MDA-MB-231 cells in a 2D colony formation assay (CFA)over a 10-day period. Our CDK4/6 degraders showed potent inhibition of cellproliferation with CFA IC50s of <50nM. We demonstrated that our CDK4/6 bifunctionaldegraders were significantly more potent in vitro than the CDK4/6i, palbociclib andribociclib, and the increased activity was due to CRBN mediated target degradation.Our CDK4/6 bifunctional degraders display excellent pharmacokinetic properties inmice with half-lives between 2-10 hours, oral bioavailability between 50-96% and arecurrently being evaluated for tumor growth inhibition against standard of caremolecules in MDA-MB-231 and MCF7 xenograft models. Clinical CDK4/6i are beingevaluated as combinations in TNBC, but the enhanced potency of CDK4/6 degradersin the TNBC cell lines shows that this approach is more promising for this aggressivetype of breast cancer. Reference: Scheidemann, Erin R, and Ayesha N Shajahan-Haq. “Resistance toCDK4/6 Inhibitors in Estrogen Receptor-Positive Breast Cancer.” International journalof molecular sciences vol. 22,22 12292. 14 Nov. 2021, doi:10.3390/ijms222212292 Citation Format: Hannah Majeski, Akinori Okano, Angela Pasis, Casey Carlson, Qiao Liu, Arvind Shakya, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. Discovery of CDK4/6 bifunctional degraders for ER+/HER2- breast cancer andtriple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1553.
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