Abstract
Relapse and refractoriness to chemotherapy may exist prior to the exposure to chemotherapeutic agents or can develop and increase during the treatment leading to acquired resistance. Despite considerable progress in developing new therapeutic agents to be administered singly or in combination with the old regimen, drug resistance poses a serious concern preventing adequate response to Acute Myeloid Leukemia (AML) treatment with cytarabine (AraC) and daunorubicin (DNR).
Differentially expression analysis of the RNA sequencing data between the naive and the AraC-resistant human monocytic MLL-AF9 cell line THP1 revealed cathepsin G (CTSG) as one of the top hits among the upregulated genes with Log2Fc 4.2. This was further validated in the AML cell line by q-RT-PCR with a fold change ~5 and immunoblot, confirming increased CTSG expression in THP1 Ara-C resistant cell line. Although the role of (CTSG) in host defence, granulopoiesis has been intensively investigated, its role in acquired cytarabine resistance has not been explored yet.
To investigate the functional role of CTSG in the acquired resistant model, we performed CRISPR/Cas9-based editing of CTSG in THP1 Ara-C resistant cell line using dual gRNAs, which resulted in a robust deletion of the targeted region and complete loss of protein expression as measured by immunoblotting. CTSG KO cells showed a reduced cell proliferation rate with a significant reduction at 72hrs (compared to the scrambled gRNA (ScrG) transduced cells doubling time CTSG KO=30.3 vs ScrG=22.39, p= 0.0049). We also checked the sensitivity of the CTSG KO cells to AraC and other chemo drugs (DNR, ATO and Venetoclax). In vitro cytotoxicity assay (72hrs) for the CTSG KO cells treated with AraC showed sensitivity to Ara-C with a reduction in IC50 as low as the parental IC50 (IC50 ScrG: 1457 µM; IC50 CTSG KO: 38.13 µM; IC50 Parental: 56.6 µM). CTSG KO cells also sensitized the cells to DNR (IC50 ScrG: 288nM vs and CTSG KO: 58nM but not to ATO and venetoclax.
Further, we measured the intracellular DNR by flow cytometry in the CTSG KO cells. There was a considerable increase in the intracellular DNR at the end of the treatment compared to the ScrG cells (~1.5-fold, p <0.0001). Our results show that CTSG KO re-sensitized the resistant cells to cytarabine and showed significant sensitivity to DNR.
To further understand the molecular mechanism contributing to the increased sensitivity of the CTSG KO cells to AraC, we checked our basal RNA sequencing data for the expression of AraC metabolizing genes. The THP1 Ara-C R cell line showed decreased expression of DCK and increased expression of NT5C2 and CDA compared to the parental cell line. We checked the RNA expression of Ara-C metabolizing genes post CTSG KO by RT-q PCR. There was a 6-fold increase in DCK and a 5-fold decrease in NT5C2 with a further increase in SAMHD1 and CDA genes with no changes in the RRM1 and ENT1 genes, suggesting that CTSG KO increased the sensitivity to by rewiring the AraC metabolic pathway. Further studies are ongoing to assess the in-vivo efficacy of molecular and pharmacological inhibition of CTSG in reversing AraC resistance in the AML mouse model.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
