LRH-1/NR5A2 interacts with the glucocorticoid receptor to regulate glucocorticoid resistance

Nuclear receptors are transcription factors with important functions in a variety of physiological and pathological processes. Targeting glucocorticoid receptor (GR) activity using glucocorticoids is a cornerstone in the treatment of patients with T cell acute lymphoblastic leukemia (T-ALL), and resistance to GC-induced cell death is associated with poor outcome and a high risk for relapse. Next to ligand-binding, heterodimerization with other transcription factors presents an important mechanism for the regulation of GR activity. Here, we describe a GC-induced direct association of the Liver Receptor Homolog-1 (LRH-1) with the GR in the nucleus, which results in reciprocal inhibition of transcriptional activity. Pharmacological and molecular interference with LRH-1 impairs proliferation and survival in T-ALL and causes a profound sensitization to GC-induced cell death, even in GC-resistant T-ALL. Our data illustrate that direct interaction between GR and LRH-1 critically regulates glucocorticoid sensitivity in T-ALL opening up new perspectives for developing innovative therapeutic approaches to treat GC-resistant T-ALL

B and T cell acute lymphoblastic leukemia evade chemotherapy at distinct sites in the bone marrow

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support treatment escape. Using 3D fluorescence imaging of 10 primary ALL xenografts we identify sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detect B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon short-term chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment

Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia

Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.Peer reviewe

LRH-1/NR5A2 interacts with the glucocorticoid receptor to regulate glucocorticoid resistance

Nuclear receptors are transcription factors with important functions in a variety of physiological and pathological processes. Targeting glucocorticoid receptor (GR) activity using glucocorticoids is a cornerstone in the treatment of patients with T cell acute lymphoblastic leukemia (T-ALL), and resistance to GC-induced cell death is associated with poor outcome and a high risk for relapse. Next to ligand-binding, heterodimerization with other transcription factors presents an important mechanism for the regulation of GR activity. Here, we describe a GC-induced direct association of the Liver Receptor Homolog-1 (LRH-1) with the GR in the nucleus, which results in reciprocal inhibition of transcriptional activity. Pharmacological and molecular interference with LRH-1 impairs proliferation and survival in T-ALL and causes a profound sensitization to GC-induced cell death, even in GC-resistant T-ALL. Our data illustrate that direct interaction between GR and LRH-1 critically regulates glucocorticoid sensitivity in T-ALL opening up new perspectives for developing innovative therapeutic approaches to treat GC-resistant T-ALL.publishe

TNFR2 is required for RIP1-dependent cell death in human 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

Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support

Interactions with the bone marrow microenvironment are essential for leukemia survival and disease progression. We developed an imaging-based RNAi platform to identify protective cues from bone marrow derived mesenchymal stromal cells (MSC) that promote survival of primary acute lymphoblastic leukemia (ALL) cells. Using a candidate gene approach, we detected distinct responses of individual ALL cases to RNA interference with stromal targets. The strongest effects were observed when interfering with solute carrier family 3 member 2 (SLC3A2) expression, which forms the cystine transporter xc − when associated with SLC7A11. Import of cystine and metabolism to cysteine by stromal cells provides the limiting substrate to generate and maintain glutathione in ALL. This metabolic interaction reduces oxidative stress in ALL cells that depend on stromal xc −. Indeed, cysteine depletion using cysteine dioxygenase resulted in leukemia cell death. Thus, functional evaluation of intercellular interactions between leukemia cells and their microenvironment identifies a selective dependency of ALL cells on stromal metabolism for a relevant subgroup of cases, providing new opportunities to develop more personalized approaches to leukemia treatment.ISSN:1949-255

Repurposing anthelmintic agents to eradicate resistant leukemia

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 anti-leukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and T-ALL 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

Activation of concurrent apoptosis and necroptosis by SMAC mimetics for the treatment of refractory and relapsed ALL

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

B- and T-cell acute lymphoblastic leukemias evade chemotherapy at distinct sites in the bone marrow

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support escape from treatment. Using three-dimensional fluorescence imaging of ten primary ALL xenografts we identified sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detected B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon short-term chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment.ISSN:0390-6078ISSN:1592-872