Phenotype-based drug screening reveals association between venetoclax response and differentiation stage in acute myeloid leukemia

Ex vivo drug testing is a promising approach to identify novel treatment strategies for acute myeloid leukemia (AML). However, accurate blast- specific drug responses cannot be measured with homogeneous "add-mix-measure" cell viability assays. In this study, we implemented a flow cytometry-based approach to simultaneously evaluate the ex vivo sensitivity of different cell populations in 34 primary AML samples to seven drugs and 27 rational drug combinations. Our data demonstrate that different cell populations present in AML samples have distinct sensitivity to targeted therapies. Particularly, blast cells of FAB M0/1 AML showed high sensitivity to venetoclax. In contrast, differentiated monocytic cells abundantly present in M4/5 subtypes showed resistance to Bcl-2 inhibition, whereas immature blasts in the same samples were sensitive, highlighting the importance of blast-specific readouts. Accordingly, in the total mononuclear cell fraction the highest BCL2/MCL1 gene expression ratio was observed in M0/1 and the lowest in M4/5 AML. Of the seven tested drugs, venetoclax had the highest blast-specific toxicity, and combining venetoclax with either MEK inhibitor trametinib or JAK inhibitor ruxolitinib effectively targeted all venetoclax-resistant blasts. In conclusion, we show that ex vivo efficacy of targeted agents and particularly Bcl-2 inhibitor venetoclax is influenced by the cell type, and accurate blast-specific drug responses can be assessed with a flow cytometry-based approach.Peer reviewe

HOX gene expression predicts response to BCL-2 inhibition in acute myeloid leukemia

Peer reviewe

Dasatinib induces fast and deep responses in newly diagnosed chronic myeloid leukaemia patients in chronic phase : clinical results from a randomised phase-2 study (NordCML006)

Peer reviewe

Early response evaluation by single cell signaling profiling in acute myeloid leukemia

Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phosphoERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.Peer reviewe

Implementing a Functional Precision Medicine Tumor Board for Acute Myeloid Leukemia

We generated ex vivo drug-response and multiomics profi ling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identifi cation of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profi ling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes signifi cant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.Peer reviewe

Implementing a Functional Precision Medicine Tumor Board for Acute Myeloid Leukemia

We generated ex vivo drug-response and multiomics profi ling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identifi cation of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profi ling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes signifi cant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.Peer reviewe

Single cell immune profiling by mass cytometry of newly diagnosed chronic phase chronic myeloid leukemia treated with nilotinib

Monitoring of single cell signal transduction in leukemic cellular subsets has been proposed to provide deeper understanding of disease biology and prognosis, but has so far not been tested in a clinical trial of targeted therapy. We developed a complete mass cytometry analysis pipeline for characterization of intracellular signal transduction patterns in the major leukocyte subsets of chronic phase chronic myeloid leukemia. Changes in phosphorylated Bcr-Abl1 and the signaling pathways involved were readily identifiable in peripheral blood single cells already within three hours of the patient receiving oral nilotinib. The signal transduction profiles of healthy donors were clearly distinct from those of the patients at diagnosis. Furthermore, using principal component analysis, we could show that phosphorylated transcription factors STAT3 (Y705) and CREB (S133) within seven days reflected BCR-ABL1(IS) at three and six months. Analyses of peripheral blood cells longitudinally collected from patients in the ENEST1st clinical trial showed that single cell mass cytometry appears to be highly suitable for future investigations addressing tyrosine kinase inhibitor dosing and effect. (clinicaltrials. gov identifier: 01061177)Peer reviewe

Expression of TP53 isoforms p53β or p53γ enhances chemosensitivity in TP53(null) cell lines

The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null) background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1)), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level