More precise treatment strategies are urgently needed to decrease toxicity and improve outcomes for treatment-refractory leukemia. We used ex vivo drug response profiling of high-risk, relapsed, or refractory acute lymphoblastic leukemia (ALL) cases and identified a subset with exquisite sensitivity to small-molecule mimetics of the second mitochondria-derived activator of caspases (SMAC) protein. Potent ex vivo activity of the SMAC mimetic (SM) birinapant correlated with marked in vivo antileukemic effects, as indicated by delayed engraftment, decreased leukemia burden, and prolonged survival of xenografted mice. Antileukemic activity was dependent on simultaneous execution of apoptosis and necroptosis, as demonstrated by functional genomic dissection with a multicolored lentiCRISPR approach to simultaneously disrupt multiple genes in patient-derived ALL. SM specifically targeted receptor-interacting protein kinase 1 (RIP1)-dependent death, and CRISPR-mediated disruption of RIP1 completely blocked SM-induced death yet had no impact on the response to standard antileukemic agents. Thus, SM compounds such as birinapant circumvent escape from apoptosis in leukemia by activating a potent dual RIP1-dependent apoptotic and necroptotic cell death, which is not exploited by current therapy. Ex vivo drug activity profiling could provide important functional diagnostic information to identify patients who may benefit from targeted treatment with birinapant in early clinical trials.
Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.
Despite major advances in the treatment of patients with acute lymphoblastic leukemia in the last decades, refractory and/or relapsed disease remains a clinical challenge, and relapsed leukemia patients have an exceedingly dismal prognosis. Dysregulation of apoptotic cell death pathways is a leading cause of drug resistance; thus, alternative cell death mechanisms, such as necroptosis, represent an appealing target for the treatment of high-risk malignancies. We and other investigators have shown that activation of receptor interacting protein kinase 1 (RIP1)–dependent apoptosis and necroptosis by second mitochondria derived activator of caspase mimetics (SMs) is an attractive antileukemic strategy not currently exploited by standard chemotherapy. However, the underlying molecular mechanisms that determine sensitivity to SMs have remained elusive. We show that tumor necrosis factor receptor 2 (TNFR2) messenger RNA expression correlates with sensitivity to SMs in primary human leukemia. Functional genetic experiments using clustered regularly interspaced short palindromic repeats/Cas9 demonstrate that TNFR2 and TNFR1, but not the ligand TNF-α, are essential for the response to SMs, revealing a ligand-independent interplay between TNFR1 and TNFR2 in the induction of RIP1-dependent cell death. Further potential TNFR ligands, such as lymphotoxins, were not required for SM sensitivity. Instead, TNFR2 promotes the formation of a RIP1/TNFR1-containing death signaling complex that induces RIP1 phosphorylation and RIP1-dependent apoptosis and necroptosis. Our data reveal an alternative paradigm for TNFR2 function in cell death signaling and provide a rationale to develop strategies for the identification of leukemias with vulnerability to RIP1-dependent cell death for tailored therapeutic interventions.
Despite rapid progress in genomic profiling in acute lymphoblastic leukemia (ALL), identification of actionable targets and prediction of response to drugs remains challenging. To identify specific vulnerabilities in ALL, we performed a drug screen using primary human ALL samples cultured in a model of the bone marrow microenvironment combined with high content image analysis. Among the 2487 FDA-approved compounds tested, anthelmintic agents of the class of macrocyclic lactones exhibited potent anti-leukemia activity, similar to the already known antileukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and TALL including refractory relapse cases confirmed strong anti-leukemia activity with IC 50 values in the low micromolar range. Anthelmintic agents increased intracellular chloride levels in primary leukemia cells, inducing mitochondrial outer membrane depolarization and cell death. Supporting the notion that simultaneously targeting cell death machineries at different angles may enhance the cell death response, combination of anthelmintic agents with the BCL-2 antagonist navitoclax or with the chemotherapeutic agent dexamethasone showed synergistic activity in primary ALL. These data reveal anti-leukemia activity of anthelmintic agents and support exploiting drug repurposing strategies to identify so far unrecognized anti-cancer agents with potential to eradicate even refractory leukemia.
CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function.However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.
The spleen tyrosine kinase (SYK) and ζ-associated protein of 70 kD (ZAP70) tyrosine kinases play critical roles in proximal signal transduction of B-cell (BCR) and T-cell receptors (TCR), respectively. The highly similar SYK and ZAP70 kinases share a common structure composed of two tandem SH2 domains and a carboxy-terminal kinase domain. A linker region, termed interdomain B, connects the SH2 domains to the kinase domain and is important for kinase activation. Despite their conserved structure, SYK and ZAP70 are expressed in a largely mutually exclusive manner and play analogous roles in BCR- and TCR-signaling. Cross-lineage activation of ZAP70 in B cells was previously identified in chronic lymphocytic leukemia (CLL), which is characterized by clonal accumulation of malignant CD5+ B-cell cells that retain dependency on the BCR for survival signals. Nearly half of CLL cases show co-expression of SYK and ZAP70, and these patients have an aggressive disease course and a poor prognosis. Our analysis shows that in addition to CLL, aberrant ZAP70 expression occurs in other B-cell malignancies, e.g. TCF3-PBX1 pre-B ALL and B-cell lymphoma subsets that depend on survival signals from a functional (pre-) BCR. These findings suggest that interactions between SYK and ZAP70 may function to fine-tune strength of oncogenic BCR-signaling. To test this hypothesis, we have used a combination of molecular and proteomic approaches. We studied mechanisms by which ZAP70 integrates into BCR-mediated signals, and how the function of ZAP70 in B-cells differs from its native role downstream of the TCR. We demonstrate that ectopically expressed SYK and ZAP70 proteins are constitutively phosphorylated in BCR-ABL1+ B-ALL cells, but these induce distinctive signaling thresholds. CRISPR-mediated deletion of SYK or ZAP70 in leukemic cells further revealed that SYK and ZAP70 regulate unique signaling pathways in B-cells. We also demonstrate that ZAP70 is activated following BCR stimulation of lymphoma cells, and SYK/ZAP70 co-expressing cells display enhanced BCR signaling. Interestingly, enhanced BCR signaling was also observed in cells engineered to express an alternative splice variant of SYK (SYK-S). This shorter isoform of SYK, lacks a 23 amino-acid insert in the interdomain-B linker region, which is also absent in ZAP70, and may define unique protein-interactions that modulate signaling outcome. To elucidate the differential interactome of SYK, SYK-S, and ZAP70 we performed proximity-dependent biotin identification (BioID) experiments in B-cells following BCR-activation to capture the core signalling networks of these kinases in leukemic cells. In addition to expected BCR components including BLNK, PTPN6 and CBL we identified novel SYK and ZAP70 associated molecules including IKZF3, LAT2 and WAS which may play important roles in the survival of BCR-dependent malignancies. Importantly our findings highlight a role for ZAP70 in oncogenic BCR-signaling and suggest that ZAP70 promotes oncogenic BCR-signaling by limiting the ability of the BCR to induce negative B-cell selection and cell death. Disclosures No relevant conflicts of interest to declare.
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