Genome-wide loss-of-function genetic screening identifies opioid receptor μ1 as a key regulator of L-asparaginase resistance in pediatric acute lymphoblastic leukemia

Abstract

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.

Methadone—Not a magic bullet in melanoma therapy

“…Whereas in various cancer types, OPRM1 expression has been associated with higher staging and/or poor outcome, our findings suggest this is not the case in melanoma. As to the effects of Met, Friesen et al had reported that apoptosis rates increased by about 50% in leukaemia and glioblastoma cell lines treated with Met combined with chemotherapeutic agents.…”

“…Based on a series of studies suggesting a role of Met as a chemotherapy sensitizer in certain cancer types, we addressed the potential/validity of this hypothesis in the setting of melanoma.…”

“…A series of studies has pointed towards an antitumoral effect of Met in various types of human cancer. In vitro data suggest that Met alone can induce apoptosis, and, when combined with chemotherapeutic agents, sensitize cancer cells for their actions, possibly in an OPRM1‐dependent fashion . Clinically, a retrospective, non‐placebo–controlled study has pointed towards a sensitizing role of Met for temozolomide (TMZ) chemotherapy in glioblastoma …”

Against Repurposing Methadone for Glioblastoma Therapy

“…The postulated ability of methadone to enhance cancer therapy [ 16 , 17 , 18 ] has received considerable attention in recent years, but remains highly controversial. Preclinical evidence for a tumoricidal methadone effect has been obtained in vitro and in mouse models for various tumor identities, such as leukemia [ 17 , 18 , 78 , 79 , 80 ], glioblastoma [ 16 , 81 , 82 ], neuroblastoma [ 83 ], and carcinoma cells [ 13 , 14 , 83 , 84 , 85 , 86 ]. These studies are summarized in Table 3 .…”

Development and Validation of a Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry Method for the Determination of Asparagine in Human Serum

“…Besides high expression and activity of ASN, host factors, especially ASNase antibody production, were also reported to be associated with the drug resistance of ASNase. However, it has been reported that the host factors would eventually result in high expression and activity of ASN [10,13,14]. erefore, to guide the decision of switching preparations or dose escalation of ASNase, the monitoring of drug concentration or therapeutic markers were necessary for ALL patients.…”