PRMT5 INHIBITION RESTARTS A PRO-APOPTOTIC PROGRAM AND CREATES VULNERABILITY TO COMBINATION TREATMENT WITH BCL-2 INHIBITOR VENETOCLAX IN MANTLE CELL LYMPHOMA
Author(s): ,
Fiona Brown
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Inah Hwang
Affiliations:
Pathology and Laboratory Medicine,Weil Cornell Medicine,New York,United States
,
Shelby Sloan
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Claire Hinterschied
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
JoBeth Helmig-Mason
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Mackenzie Long
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Youssef Youssef
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Wing Chan
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Alexander Prouty
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Jihyun Chung
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Yang Zhang
Affiliations:
Prelude Therapeutics,Wilmington,United States
,
Kris Vaddi
Affiliations:
Prelude Therapeutics,Wilmington,United States
,
Selina Chen-Kiang
Affiliations:
Pathology and Laboratory Medicine,Weil Cornell Medicine,New York,United States
,
Maurizio DiLiberto
Affiliations:
Pathology and Laboratory Medicine,Weil Cornell Medicine,New York,United States
,
Oliver Elemento
Affiliations:
Physiology and Biophysics,Weil Cornell Medicine,New York,United States
,
Lalit Sehgal
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Lapo Alinari
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Peggy Scherle
Affiliations:
Prelude Therapeutics,Wilmington,United States
,
Rosa Lapalombella
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
,
Jihye Paik
Affiliations:
Pathology and Laboratory Medicine,Weil Cornell Medicine,New York,United States
Robert Baiocchi
Affiliations:
Internal Medicine, Division of Hematology,The Ohio State University,Columbus,United States
EHA Library. Brown F. 06/09/21; 325619; EP861
Fiona Brown
Fiona Brown
Contributions
Abstract
Presentation during EHA2021: All e-poster 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: EP861

Type: E-Poster Presentation

Session title: Lymphoma Biology & Translational Research

Background

Mantle cell lymphoma (MCL) is an incurable B cell malignancy comprising 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 4-6 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life.


Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting cell cycle progression, survival, and drug resistance. While we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models, we have seen resistance and relapse.


With PRMT5i we have seen a decrease in AKT activity which in turn releases FOXO1 from a phosphorylated, inactive form. Performing ChIP seq on FOXO1 resulted in over 800 target genes likely to be regulated by this transcription factor. Among these genes, pro-apoptotic BCL2 family proteins were seen as a potential indicators of a change of pro-apoptotic threshold with PRMT5i. This prompted us to pursue co-inhibition of PRMT5 and BCL as a synergistic treatment in MCL. 

Aims

To validate PRMT5 and BCL2 inhibition as a synergistic combination strategy in in vitro and in vivo models of MCL.


To confirm the role of FOXO1 and the BCL2 family proteins in any seen synergy.

Methods

Nine MCL cell lines were treated with PRMT5 inhibitor PRT382, at their IC50 at day nine and collected on day six. Immunoblots and rtPCR were used to look at changes in protein and transcript levels. Cells were treated alone and in combination with venetoclax. Synergy was calculated with an MTS read out on day nine and analyzed with Combenefit. shRNA clones were generated with a lentiviral transfection and viability was measured with annexin V/PI flow cytometry. Two murine paitient derived xenograft models, PDX.AA.MCL and PDX.IR.96069 were engrafted via tail vein in NSG mice. Disease burden was monitored weekly via flow cytometry.

Results

Eight of the nine MCL reached an IC50 of <1uM after nine days of PRT382 treatment. Collected samples showed an upregulation of BAX, BAK1, BBC3, and BMF in at least one cell line on a transcript and protein level. BAX was found to be the most ubiquitously upregulated. While only four cell lines were found to be sensitive to venetoclax treatment, seven showed synergy when treated with both drugs. Cell death was confirmed to be intrinsic and synergy was found to be dependent on BAX expression. Combination treatment with well-tolerated doses of PRT382 and venetoclax in the MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. Both PDX models showed an extension of life with combination treatment (P<0.001) and delayed disease progression (P<0.05).

Conclusion

This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting.

Keyword(s): Apoptosis, Methylation, Non-Hodgkin's lymphoma, Synergy

Presentation during EHA2021: All e-poster 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: EP861

Type: E-Poster Presentation

Session title: Lymphoma Biology & Translational Research

Background

Mantle cell lymphoma (MCL) is an incurable B cell malignancy comprising 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 4-6 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life.


Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting cell cycle progression, survival, and drug resistance. While we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models, we have seen resistance and relapse.


With PRMT5i we have seen a decrease in AKT activity which in turn releases FOXO1 from a phosphorylated, inactive form. Performing ChIP seq on FOXO1 resulted in over 800 target genes likely to be regulated by this transcription factor. Among these genes, pro-apoptotic BCL2 family proteins were seen as a potential indicators of a change of pro-apoptotic threshold with PRMT5i. This prompted us to pursue co-inhibition of PRMT5 and BCL as a synergistic treatment in MCL. 

Aims

To validate PRMT5 and BCL2 inhibition as a synergistic combination strategy in in vitro and in vivo models of MCL.


To confirm the role of FOXO1 and the BCL2 family proteins in any seen synergy.

Methods

Nine MCL cell lines were treated with PRMT5 inhibitor PRT382, at their IC50 at day nine and collected on day six. Immunoblots and rtPCR were used to look at changes in protein and transcript levels. Cells were treated alone and in combination with venetoclax. Synergy was calculated with an MTS read out on day nine and analyzed with Combenefit. shRNA clones were generated with a lentiviral transfection and viability was measured with annexin V/PI flow cytometry. Two murine paitient derived xenograft models, PDX.AA.MCL and PDX.IR.96069 were engrafted via tail vein in NSG mice. Disease burden was monitored weekly via flow cytometry.

Results

Eight of the nine MCL reached an IC50 of <1uM after nine days of PRT382 treatment. Collected samples showed an upregulation of BAX, BAK1, BBC3, and BMF in at least one cell line on a transcript and protein level. BAX was found to be the most ubiquitously upregulated. While only four cell lines were found to be sensitive to venetoclax treatment, seven showed synergy when treated with both drugs. Cell death was confirmed to be intrinsic and synergy was found to be dependent on BAX expression. Combination treatment with well-tolerated doses of PRT382 and venetoclax in the MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. Both PDX models showed an extension of life with combination treatment (P<0.001) and delayed disease progression (P<0.05).

Conclusion

This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting.

Keyword(s): Apoptosis, Methylation, Non-Hodgkin's lymphoma, Synergy

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