DISCOVERY OF A SELECTIVE INHIBITOR OF THE SETD2 HISTONE METHYLTRANSFERASE WITH POTENT IN VITRO AND IN VIVO ACTIVITY IN PRECLINICAL MODELS OF MULTIPLE MYELOMA
Author(s): ,
Jennifer Totman
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
John W. Lampe
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Thomas V. Riera
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Cuyue Tang
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Dorothy Brach
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Vinny Motwani
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Joshua S. Alford
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Neil A. Farrow
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Daniel T. Dransfield
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Kenneth W. Duncan
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Emily Deutschman
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Sean T. Eckley
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Selene Howe
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Alejandra Raimondi
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Michael J. Thomenius
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
,
Jeffery L. Kutok
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
Kat Cosmopoulos
Affiliations:
Research,Epizyme, Inc.,Cambridge,United States
EHA Library. Totman J. 06/09/21; 324584; S176
Jennifer Totman
Jennifer Totman
Contributions
Abstract
Presentation during EHA2021: All Oral presentations will be made available as of Friday, June 11, 2021 (09:00 CEST) and will be accessible for on-demand viewing until August 15, 2021 on the Virtual Congress platform.

Abstract: S176

Type: Oral Presentation

Session title: Basic and translational myeloma research

Background
Multiple myeloma (MM) is a heterogeneous disease of monoclonal plasma cells. Nearly 50% of MM patients harbor translocations of the immunoglobulin heavy chain (IgH) gene on chromosome 14q32 leading to overexpression of putative oncogenes (Kuehl, 2002).  One of the most common translocations in MM (15-20%) juxtaposes IgH control elements with two genes on chromosome 4, fibroblast growth factor receptor 3 and multiple myeloma SET domain (MMSET). This (4;14) (p16; q32) translocation is associated with poor prognosis and less effective treatment with standard of care (SOC) therapy. MMSET expression has been confirmed as a driver in t(4;14) MM pathogenesis (Keats, 2003), but MMSET remains undruggable. SETD2 is currently the only known histone methyltransferase (HMT) capable of catalyzing H3K36 trimethylation, utilizing MMSET-catalyzed H3K36me2 as its substrate. To date, a gain of function role for SETD2 in oncogenesis and tumor survival has not been demonstrated. Our data suggests that the overexpression of MMSET and subsequent increase in H3K36me2 in t(4;14) MM leads to a dependency on SETD2 for MM cell growth and survival.

Aims
Characterization of a small molecule SETD2 inhibitor exhibiting potent in vitro anti-proliferative activity and a pharmacokinetic profile that supports in vivo efficacy in preclinical models of MM.

Methods
Biochemical selectivity assays performed in a panel of HMTs and a 14-day cellular proliferation assay determined IC50’s in MM cell lines of diverse genetic subtypes. Combinatorial potential of SETD2 inhibition with anti-MM SOC agents was evaluated in 7-day cotreatment in vitro cellular assays. Mouse xenograft studies in MM models assessed tumor growth inhibition. H3K36me3 levels determined by western blot and ELISA to evaluate target engagement by SETD2 inhibitor. 

Results
A small molecule inhibitor of SETD2 methyltransferase enzymatic activity was characterized demonstrating an IC50 value of 5.3 nM in biochemical assays with greater than 10,000-fold selectivity against a panel of 14 HMTs. Inhibition of SETD2 results in potent inhibition of proliferation in 6 of 7 t(4;14) MM cell lines in vitro with an average IC50  of 0.14 uM and resulted in marked reduction of H3K36me3. Isogenic t(4;14) cell lines containing allele-specific knock-out of MMSET demonstrated sensitivity to SETD2 inhibition only in the presence of the overexpressed MMSET allele. In vivo efficacy studies in the KMS11 t(4;14) cell line-derived xenograft model resulted in robust tumor growth inhibition (TGI) in a dose-dependent manner with maximal TGI of 95% achieved at the top doses administered and corresponding reduction in intratumoral H3K36me3 levels. Interestingly, in vitro anti-proliferative activity was also demonstrated in 13 of 15 non-t(4;14) MM cell lines tested with an average IC50 of 1.8 uM. SETD2 inhibition in xenografts from the two most sensitive non-t(4;14) MM cell lines resulted in strong TGI with maximal inhibition of 92% at the top dose administered. Lastly, in vitro synergy was observed with MM SOCs, supporting a rationale to combine SETD2 inhibition with current MM therapies.

Conclusion
To our knowledge, these are the first preclinical data to show successful targeting of the underlying oncogenic mechanism driven by MMSET overexpression in t(4;14) MM and the first demonstration of SETD2 as an oncogenic driver. Importantly, this work validates the targeting of SETD2 with a small molecule inhibitor in MM and presents a promising potential therapeutic option for the high-risk t(4;14) MM patient population.

Keyword(s): Hypermethylation, IgH translocation, Multiple myeloma, Oncogene

Presentation during EHA2021: All Oral presentations will be made available as of Friday, June 11, 2021 (09:00 CEST) and will be accessible for on-demand viewing until August 15, 2021 on the Virtual Congress platform.

Abstract: S176

Type: Oral Presentation

Session title: Basic and translational myeloma research

Background
Multiple myeloma (MM) is a heterogeneous disease of monoclonal plasma cells. Nearly 50% of MM patients harbor translocations of the immunoglobulin heavy chain (IgH) gene on chromosome 14q32 leading to overexpression of putative oncogenes (Kuehl, 2002).  One of the most common translocations in MM (15-20%) juxtaposes IgH control elements with two genes on chromosome 4, fibroblast growth factor receptor 3 and multiple myeloma SET domain (MMSET). This (4;14) (p16; q32) translocation is associated with poor prognosis and less effective treatment with standard of care (SOC) therapy. MMSET expression has been confirmed as a driver in t(4;14) MM pathogenesis (Keats, 2003), but MMSET remains undruggable. SETD2 is currently the only known histone methyltransferase (HMT) capable of catalyzing H3K36 trimethylation, utilizing MMSET-catalyzed H3K36me2 as its substrate. To date, a gain of function role for SETD2 in oncogenesis and tumor survival has not been demonstrated. Our data suggests that the overexpression of MMSET and subsequent increase in H3K36me2 in t(4;14) MM leads to a dependency on SETD2 for MM cell growth and survival.

Aims
Characterization of a small molecule SETD2 inhibitor exhibiting potent in vitro anti-proliferative activity and a pharmacokinetic profile that supports in vivo efficacy in preclinical models of MM.

Methods
Biochemical selectivity assays performed in a panel of HMTs and a 14-day cellular proliferation assay determined IC50’s in MM cell lines of diverse genetic subtypes. Combinatorial potential of SETD2 inhibition with anti-MM SOC agents was evaluated in 7-day cotreatment in vitro cellular assays. Mouse xenograft studies in MM models assessed tumor growth inhibition. H3K36me3 levels determined by western blot and ELISA to evaluate target engagement by SETD2 inhibitor. 

Results
A small molecule inhibitor of SETD2 methyltransferase enzymatic activity was characterized demonstrating an IC50 value of 5.3 nM in biochemical assays with greater than 10,000-fold selectivity against a panel of 14 HMTs. Inhibition of SETD2 results in potent inhibition of proliferation in 6 of 7 t(4;14) MM cell lines in vitro with an average IC50  of 0.14 uM and resulted in marked reduction of H3K36me3. Isogenic t(4;14) cell lines containing allele-specific knock-out of MMSET demonstrated sensitivity to SETD2 inhibition only in the presence of the overexpressed MMSET allele. In vivo efficacy studies in the KMS11 t(4;14) cell line-derived xenograft model resulted in robust tumor growth inhibition (TGI) in a dose-dependent manner with maximal TGI of 95% achieved at the top doses administered and corresponding reduction in intratumoral H3K36me3 levels. Interestingly, in vitro anti-proliferative activity was also demonstrated in 13 of 15 non-t(4;14) MM cell lines tested with an average IC50 of 1.8 uM. SETD2 inhibition in xenografts from the two most sensitive non-t(4;14) MM cell lines resulted in strong TGI with maximal inhibition of 92% at the top dose administered. Lastly, in vitro synergy was observed with MM SOCs, supporting a rationale to combine SETD2 inhibition with current MM therapies.

Conclusion
To our knowledge, these are the first preclinical data to show successful targeting of the underlying oncogenic mechanism driven by MMSET overexpression in t(4;14) MM and the first demonstration of SETD2 as an oncogenic driver. Importantly, this work validates the targeting of SETD2 with a small molecule inhibitor in MM and presents a promising potential therapeutic option for the high-risk t(4;14) MM patient population.

Keyword(s): Hypermethylation, IgH translocation, Multiple myeloma, Oncogene

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