Samanta Kisele

Effect of in vivo treatment on mouse weight.

Figure S1 from Evaluation of the Role of AXL in Fusion-positive Pediatric Rhabdomyosarcoma Identifies the Small-molecule Inhibitor Bemcentinib (BGB324) as Potent Chemosensitizer

Supplementary Figures and Tables

AXL genetic removal and pathway activation.

Effect of the small-molecule AXL inhibitors.

Combination of chemotherapy and AXL inhibitors.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive lymphoid malignancy with limited treatment options. To discover new treatment targets for T-PLL, we performed high-throughput drug sensitivity screening on 30 primary patient samples ex vivo. After screening >2800 unique compounds, we found T-PLL to be more resistant to most drug classes, including chemotherapeutics, than other blood cancers. Furthermore, we discovered previously unreported vulnerabilities of T-PLL. T-PLL cells exhibited a particular sensitivity to drugs targeting autophagy (thapsigargin and bafilomycin A1), nuclear export (selinexor), and inhibitor of apoptosis proteins (IAPs; birinapant), sensitivities that were also shared by other T-cell malignancies. Through bulk and single-cell RNA sequencing, we found these compounds to activate the Toll-like receptor (bafilomycin A1), p53 (selinexor), and tumor necrosis factor α (TNF-α)/NF-κB signaling pathways (birinapant) in T-PLL cells. Focusing on birinapant for its potential in drug repurposing, we uncovered that IAP inhibitor–induced cell death was primarily necroptotic and dependent on TNF-α. Through spectral flow cytometry, we confirmed the absence of cleaved caspase-3 in IAP inhibitor–treated T-PLL cells and show that IAP inhibition reduces the proliferation of T-PLL cells stimulated ex vivo, while showing only a limited effect on nonmalignant T-cells. In summary, our study maps the drug sensitivity of T-PLL across a broad range of targets and identifies new therapeutic approaches for T-PLL by targeting IAPs, exportin 1, and autophagy, highlighting potential candidates for drug repurposing and novel treatment strategies.

Insufficient eradication of cancer cells and survival of drug tolerant clones are major relapse driving forces. Underlying molecular mechanisms comprise activated pro-survival and anti-apoptotic signaling leading to insufficient apoptosis and drug resistance. The identification of programmed cell death pathways alternative to apoptosis opens up for possibilities to antagonize apoptosis escape routes. We have earlier shown that acute lymphoblastic leukemia (ALL) harbours a distinct propensity to undergo cell death by RIPK1-dependent necroptosis, activated by small molecule second mitochondria-derived activators of caspase (SMAC) mimetics. Despite demonstrated safety and tolerability of SMAC mimetics in clinical trials, their efficacy as single agent appears still limited, highlighting the need for combinatorial treatments. Here, we investigate so far unexplored regulatory mechanisms of necroptosis and identify targets for interference to augment the necroptotic anti-leukemia response. Ex vivo drug response profiling in a model of the bone marrow microenvironment reveals powerful synergy of necroptosis induction with histone deacetylase inhibition. Subsequent transcriptome analysis and functional in vivo CRISPR screening identify gene regulatory circuitries through the master transcription regulators SP1, p300 and HDAC2 to drive necroptosis. While deletion of SP1 or p300 confers resistance to necroptosis, loss of HDAC2 sensitizes to RIPK1-dependent cell death by SMAC mimetics. Consequently, our data inform strong in vivo anti-leukemic activity of combinatorial necroptosis induction and HDAC inhibition in patient-derived human leukemia models. Thus, transcriptional dependency of necroptosis activation is a key regulatory mechanism that identifies novel targets for interference, pointing out a strategy to exploit alternative non-apoptotic cell death pathways to eradicate resistant disease.

Effect of the small-molecule AXL inhibitors.

Effect of in vivo treatment on mouse weight.