Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMA) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk ...myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5-azacytidine (5-AZA) could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in N=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene expression of the pro-apoptotic factor NOXA in ASXL1-mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.
Hypomethylating therapy with 5-azacytidine (5-Aza) is a standard-of-care for patients with higher-risk myelodysplastic syndromes (MDS). Response is induced in approximately 50% of 5-Aza treated ...patients. However, despite robust efficacy in responders, relapse is almost certain. Recently, inhibitors of anti-apoptotic BCL-2 protein family members have shown potent activity against AML and higher-risk MDS in combination with 5-Aza. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has shown anti-leukemic effects in combination with 5-Aza in a phase 1 study of AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). Additionally, Alv has entered a phase 1b/2 study in patients with higher-risk MDS (NCT03593915). In order to possibly identify biomarkers of response, we performed a comprehensive pre-clinical in vitro assessment of Alv combined with 5-Aza using a clinically well-characterized cohort of n=40 MDS (high risk) patients and n=11 healthy controls.
CD34+ HSCs were purified from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of healthy controls were obtained from femur head replacement surgery bone specimens. Hematopoietic stem cells (HSCs) were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-Aza for 48h, Alv for 24h or their sequential combination (5-Aza for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo and Annexin-V apoptosis assays. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes.
The combination of 5-Aza+Alv showed an additive cytotoxic effect on CD34+ MDS cells in CellTiter- Glo cell viability assays (median cell viability = 74%, 73.8% and 55% for 5-Aza, Alv and combination respectively, p<0.0001). In annexin-V apoptotic assay, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to healthy CD34+ cells (median % of apoptotic and dead cells = 36.6% for MDS vs 25.6% for healthy group, p=0.0288). Of note, the presence of ASXL1 and ZRSR2 mutations was associated with higher cytotoxic activity of 5-Aza+Alv combination. In particular, ZRSR2 mutations had an independent impact on the cell viability in a multivariable analysis (p=0.035). Overall, we provided pre-clinical support for the use of 5-Aza+Alv combination for higher risks MDS and identified ASXL1 and ZRSR2 mutations as potential genetic biomarkers of response.
Schmitt: Affimed GmbH: Research Funding. Jawhar: Celgene: Other: Travel support; Takeda: Honoraria, Other: Travel support; Stemline: Consultancy, Honoraria; Blueprint Medicines: Honoraria; Novartis: Consultancy, Honoraria, Other: Travel support, Speakers Bureau. Foulks: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2. Hofmann: Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Nowak: Celgene: Honoraria; Takeda: Honoraria; Affimed: Research Funding; Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding.
Abstract
The hypomethylating agent 5-azacytidine (5-Aza) is a standard-of-care for patients with higher-risk myelodysplastic syndromes (MDS). Although an initial response is induced in approximately ...50% of 5-Aza treated patients, subsequent relapse is almost certain. Recently, inhibitors of anti-apoptotic BCL-2 protein family members have shown therapeutic potential in acute myeloid leukemia (AML) and higher-risk MDS. Alvocidib (Alv), a CDK9 inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has shown anti-leukemic effects in a phase 1 study of patients with AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and 5-Aza or decitabine in higher-risk patients with MDS was recently completed (NCT03593915); however, biomarkers for response to the Alv and 5-Aza combination are not well characterized. To identify potential biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-Aza combination using a clinically well-characterized cohort of n=45 higher-risk patients with MDS and n=11 healthy controls (HC). CD34+ cells were purified from bone marrow (BM) aspirates using positive selection with MACS beads. CD34+ cells of HC were obtained from femur head replacement surgery bone specimens. After 4 days of expansion in SFEM II medium containing StemSpan Myeloid Expansion Supplement, cells were treated with 5-Aza for 48h, Alv for 24h or their combination (5-Aza for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of MDS samples was assessed using MS1 peptide-based assay. Recurrent myeloid neoplasia mutations in 67 genes were assessed in BM mononuclear cells using NGS panel deep sequencing. The combination of 5-Aza+Alv had an additive cytotoxic effect on CD34+ MDS cells in CTG assay (median cell viability = 74%, 73.8% and 55% for 5-Aza, Alv and combination respectively, p<0.0001). In Annexin-V apoptosis assay, MDS samples were more sensitive to the combination treatment compared to HC (median % of apoptotic and dead cells = 36.6% for MDS vs 25.6% for HC, p=0.0288). MCL-1 dependency inversely and not significantly correlated with CD34+ cell viability in CTG assays (Spearman r=-0.37, p=0.1119). In contrast, we found significant associations between ASXL1 and ZRSR2 mutations and higher sensitivity of MDS samples to 5-Aza+Alv combination (p=0.008 and p=0.0005 in univariable analysis respectively). ZRSR2 mutations also retained an independent impact on cell viability in multivariable analysis (p=0.035). Overall, we provide pre-clinical support for the use of 5-Aza+Alv combination for higher-risk MDS and identified ASXL1 and ZRSR2 mutations as potential genetic biomarkers of augmented response.
Citation Format: Vladimir Ryabov, Nanni Schmitt, Qingyu Xu, Alexander Streuer, Johann-Christoph Jann, Alina Wein, Eva Altrock, Verena Nowak, Nadine Weimer, Julia Obländer, Iris Palme, Ahmed Jawhar, Ali Darwich, Patrick Wuchter, Christel Weiss, Georgia Metzgeroth, Jason M. Foulks, Laurenz Steiner, Mohamad Jawhar, Wolf-Karsten Hofmann, Daniel Nowak. Mutations in the ASXL1 and ZRSR2 genes are associated with the response to the combination of alvocidib and 5-azacytidine in higher-risk myelodysplastic syndromes abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6257.
Comprehending the interplay between spatial and temporal characteristics of neural dynamics can contribute to our understanding of information processing in the human brain. Graph neural networks ...(GNNs) provide a new possibility to interpret graph structured signals like those observed in complex brain networks. In our study we compare different spatio-temporal GNN architectures and study their ability to model neural activity distributions obtained in functional MRI (fMRI) studies. We evaluate the performance of the GNN models on a variety of scenarios in MRI studies and also compare it to a VAR model, which is currently often used for directed functional connectivity analysis. We show that by learning localized functional interactions on the anatomical substrate, GNN based approaches are able to robustly scale to large network studies, even when available data are scarce. By including anatomical connectivity as the physical substrate for information propagation, such GNNs also provide a multi-modal perspective on directed connectivity analysis, offering a novel possibility to investigate the spatio-temporal dynamics in brain networks.
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