Contributions
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
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