Genomics of fetal haemoglobin: a targeted approach for reticulocyte transcriptome study.

Background: Fetal haemoglobin (HbF) remains a major sickle cell disease modifier. The mechanism of HbF synthesis has been studied for several decades with the intention of increasing interventions for sickle cell disease (SCD), including drugs. However, the complex mechanism of HbF synthesis is influenced by multiple genetic factors interacting with environmental factors. In order to capture useful genetic information, especially with limited resources, one has to carefully design the study. This includes choosing the relevant participants, the correct phenotyping, the choice of samples, and the right genomic assays. This paper describes the approach undertaken as part of preparations for a reticulocyte transcriptome study intended to discover genes associated with HbF decline in newborns in Tanzania. Results: Of the 152 newborns enrolled in the larger study, 40 babies were selected for the reticulocyte transcriptome study based on their HbF levels at birth and later stage of life. Of these, 30 individuals were included under the category of high HbF levels ranging from 72.6-90% and the remaining 10 under the category of low HbF levels ranging from 5.9 - 10.3%. The reticulocyte enrichment recovery purity ranged from 85% - 97%. The total RNA concentrations obtained were >250 ng total RNA, with the average purity of 1.9 (A 260/280) respectively. The total concentration obtained was sufficient for the transcriptome and other downstream assays. Conclusion: We have documented important steps and factors to consider in identifying the relevant participants and required laboratory sample processes prior to the final stage, which involves total reticulocyte RNA sequencing.

Exploiting the Therapeutic Interaction of WNT Pathway Activation and Asparaginase for Colorectal Cancer Therapy.

Colorectal cancer is driven by mutations that activate canonical WNT/ß-catenin signaling, but inhibiting WNT has significant on-target toxicity, and there are no approved therapies targeting dominant oncogenic drivers. We recently found that activating a ß-catenin-independent branch of WNT signaling that inhibits GSK3-dependent protein degradation induces asparaginase sensitivity in drug-resistant leukemias. To test predictions from our model, we turned to colorectal cancer because these cancers can have WNT-activating mutations that function either upstream (i.e., R-spondin fusions) or downstream (APC or ß-catenin mutations) of GSK3, thus allowing WNT/ß-catenin and WNT-induced asparaginase sensitivity to be unlinked genetically. We found that asparaginase had little efficacy in APC or ß-catenin-mutant colorectal cancer, but was profoundly toxic in the setting of R-spondin fusions. Pharmacologic GSK3α inhibition was sufficient for asparaginase sensitization in APC or ß-catenin-mutant colorectal cancer, but not in normal intestinal progenitors. Our findings demonstrate that WNT-induced therapeutic vulnerabilities can be exploited for colorectal cancer therapy. SIGNIFICANCE: Solid tumors are thought to be asparaginase-resistant via de novo asparagine synthesis. In leukemia, GSK3α-dependent protein degradation, a catabolic amino acid source, mediates asparaginase resistance. We found that asparaginase is profoundly toxic to colorectal cancers with WNT-activating mutations that inhibit GSK3. Aberrant WNT activation can provide a therapeutic vulnerability in colorectal cancer.See related commentary by Davidsen and Sullivan, p. 1632.This article is highlighted in the In This Issue feature, p. 1611.

Synthetic Lethality of Wnt Pathway Activation and Asparaginase in Drug-Resistant Acute Leukemias.

Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits glycogen synthase kinase 3 (GSK3)-dependent protein ubiquitination and proteasomal degradation, a catabolic source of asparagine. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3α inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase.

PRC2 loss induces chemoresistance by repressing apoptosis in T cell acute lymphoblastic leukemia.

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming. Here, we report that mitochondrial apoptosis resistance in T cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2). In T-ALL clinical specimens, loss-of-function mutations of PRC2 core components (EZH2, EED, or SUZ12) were associated with mitochondrial apoptosis resistance. In T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action. PRC2 loss induced apoptosis resistance via transcriptional up-regulation of the LIM domain transcription factor CRIP2 and downstream up-regulation of the mitochondrial chaperone TRAP1 These findings demonstrate the importance of mitochondrial apoptotic priming as a prognostic factor in T-ALL and implicate mitochondrial chaperone function as a molecular determinant of chemotherapy response.