L-asparaginase is a critical chemotherapeutic agent for acute lymphoblastic leukemia (ALL). It hydrolyzes plasma asparagine into aspartate and NH3, causing asparagine deficit and inhibition of protein synthesis and eventually, leukemic cell death. However, patient relapse often occurs due to development of resistance. The molecular mechanism by which ALL cells acquire resistance to L-asparaginase is unknown. Therefore, we sought to identify genes that are involved in L-asparaginase resistance in primary leukemic cells. By unbiased genome-wide RNAi screening, we found that among 10 resistant ALL clones, six hits were for opioid receptor mu 1 (oprm1), two hits were for carbonic anhydrase 1 (ca1) and another two hits were for ubiquitin-conjugating enzyme E2C (ube2c). We also found that OPRM1 is expressed in all leukemic cells tested. Specific knockdown of OPRM1 confers L-asparaginase resistance, validating our genome-wide retroviral shRNA library screening data. Methadone, an agonist of OPRM1, enhances the sensitivity of parental leukemic cells, but not OPRM1-depleted cells, to L-asparaginase treatment, indicating that OPRM1 is required for the synergistic action of L-asparaginase and methadone, and that OPRM1 loss promotes leukemic cell survival likely through downregulation of the OPRM1-mediated apoptotic pathway. Consistent with this premise, patient leukemic cells with relatively high levels of OPRM1 are more sensitive to L-asparaginase treatment compared to OPRM1-depleted leukemic cells, further indicating that OPRM1 loss has a crucial role in L-asparaginase resistance in leukemic patients. Thus, our study demonstrates for the first time, a novel OPRM1-mediated mechanism for L-asparaginase resistance in ALL, and identifies OPRM1 as a functional biomarker for defining high-risk subpopulations and for the detection of evolving resistant clones. Oprm1 may also be utilized for effective treatment of L-asparaginase-resistant ALL.

l-Asparaginase (l-ASNase) is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse because of the development of resistance, but the underlying mechanism of ALL cell resistance to l-asparaginase remains unknown. Through unbiased genome-wide RNA interference screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca2+ release upon stimulation with inositol 1,4,5-triphosphate3. Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspase-3 and caspase-12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents l-asparaginase–induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to l-asparaginase, indicating the clinical relevance of HAP1 loss in the development of l-asparaginase resistance, and pointing to HAP1 as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.

Imbalance between mtHsp40 and mtHsp70 enhances Opa1L cleavage, leading to cristae remodeling and eventual mitochondrial fragmentation and defective OXPHOS. This is important for understanding functional links between chaperone activity of mtHsp40/mtHsp70 and mitochondrial biology at the molecular and cellular levels.

CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2) or an alternatively spliced variant form (hCDK5RAP2-V1) that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species.

ATRT of the CNS constitute a group of rare and aggressive early childhood tumors with poor prognosis. While there are differing chemotherapeutic regimens for ATRT, high-dose MTX is a crucial component of many therapeutic protocols. Currently, the biological mechanisms contributing to the generation of MTX resistance in ATRT are unknown. To identify genes involved in MTX resistance in ATRT, an unbiased genome-wide RNAi screen on ATRT cell lines was conducted using 24,000 distinct shRNAs covering 8,000 genes. ATRT cells were transfected with a retrovirus containing pRS-shRNA vectors and treated with puromycin for selection. The resulting cells were treated with MTX to identify resistant clones and resistant colonies were then isolated and amplified individually. Presence of shRNA inserts in each colony was determined by PCR using pRS forward and reverse primers. PCR products within each of the three resistant colonies were sequenced, leading to the identification of three distinct genes, TGIF1, HIF3A and PGAM2, as potential indicators of resistance. Western blotting verified depletion of these proteins in their respective colonies. Proliferation assays were then conducted on cells from each resistant colony alongside control cells to confirm that the identified drivers conferred resistance. Sensitivity to MTX was significantly lower in TGIF1-depleted (IC50=212±8.48nM, n=3), HIF3A-depleted (IC50=52±4.68nM, n=3) and PGAM2-depleted (IC50=41±4.13nM, n=3) cells compared to control cells (IC50=19±2.87nM, n=3), (p<0.001). In addition, more than 60% of TGIF1, HIF3A, and PGAM2-depleted cells survived the maximum MTX treatment (100nM), while less than 20% of control cells survived this treatment. Our study using an unbiased genome-wide RNAi screen approach has shown that depletion of TGIF1, HIF3A and PGAM2 are potential molecular markers of MTX resistance in ATRT. Screening for their occurrence may help to identify patients at high risk of MTX resistance and may also serve as targets for future novel therapeutics development.

Introduction: Epigenetic alterations leading to the silencing of key tumor suppressor genes by promoter hypermethylation have been implicated in the pathogenesis of a number of malignancies, including MDS and AML. Currently, the prototypical DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (decitabine) has been studied in a number of diverse protocols as an anti-leukemic agent. However, the pattern of non-responsiveness to decitabine appears to be complex and multifactorial with some patients showing primary resistance whereas others develop resistance following initial responsiveness. To further understand the molecular mechanisms that define growth regulatory networks in pediatric AML, we have established and performed initial characterization using primary blasts that showed increased cell survival and proliferation in the presence of decitabine. Methods: Bone marrow leukemic blasts from a relapsed pediatric AML patient, who received only conventional chemotherapy, were obtained following local REB approval and informed parental consent. Upon in vitro culture to identify effective therapeutic agents, enhanced cell survival was noted in wells containing decitabine (1uM) compared to untreated cells. This cell population was further expanded in higher concentrations of decitabine that tolerated concentrations higher than 10 uM. These cells were then clonally expanded, and the resulting cell line designated POETIC1, was screened in growth inhibition assays against a panel of 142 pharmaceutical pipe-line agents that target known growth regulatory pathways and signaling molecules. The original primary leukemic cells and normal lymphocytes were used as control. Gene expression analyses were carried out using humanHT-12 v4 Expression BeadChip whole-genome expression arrays, normalized and analyzed using the Illumina BeadStudio Software. The distribution and plasticity, and quantity of DNA methylation were studied using the Illumina Infinium Human Methylation BeadChip Assay. Results: POETIC1 cells showed a differential drug sensitivity in approximately 20% of the agents tested. This includes enhanced susceptibility to agents that interfere with cell cycle regulation such as aurora kinase inhibitors, PLK, HDAC inhibitors and agents that targeted mTOR and proteasome activities. Transcriptome profiling revealed that 399 genes were down-regulated and 977 were up-regulated in the leukemia cells, compared to normal controls. POETIC 1 cells had significantly up-regulated DNA repair, cell cycle, oxidative phosphorylation and many other pro-survival pathways. Pathway analysis revealed that up-regulated genes belonged to cell cycle control and pro-survival signalling pathways including genes encoding for cyclins A and B, Cdc 7, Cdc 20 among others. They also had down-regulated genes relating to apoptosis, endocytosis and cell differentiation pathways. Global DNA methylome analysis revealed profound genome-wide deregulation of DNA methylation in POETIC 1 cells with a large number of genes were differentially methylated, including those involved in the control of cell cycle, oxidative phosphorylation, apoptosis and DNA repair pathways. Discussion: Our findings indicate aberrant cell cycle and metabolic pathways in leukemia cells with primary resistance to decitabine. The POETIC1 cell line, provides a critical experimental tool to investigate the role of epigenetic alterations in leukemogenesis as well as the molecular and physiological mechanisms that define primary resistance to methyltransferase inhibitors and facilitate the identification of novel therapeutic agents for refractory disease in future clinical studies. Disclosures No relevant conflicts of interest to declare.