Lentiviral-based mutagenesis to identify mutations that confer resistance to anti-cancer drugs [lenti]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE164663 | GEO | 2021/07/25
REPOSITORIES: GEO
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Project description:Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT) and we show this property can be leveraged to identify mutations that confer resistance to targeted anti-cancer drugs, a technique we term “LentiMutate”. First, we improved LentiMutate by making the lentiviral RT more error-prone. Next, we applied this technique to two anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL that confer resistance to BCR-ABL inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development
2021-07-25 | GSE164661 | GEO
Project description:Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). We optimized and leveraged this property to identify drug resistance mutations, a technique we term LentiMutate. After validating this technique by identifying clinically relevant EGFR resistance mutations, we applied this technique to two additional anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL1 that confer resistance to BCR-ABL1 inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development.
2021-07-28 | GSE180965 | GEO
Project description:MiR-142 is dynamically expressed and plays a regulatory role in hematopoiesis. Based on the simple observation that miR-142 levels are significantly lower in CD34+CD38- cells from blast crisis (BC) chronic myeloid leukemia (CML). CML patients compared with chronic phase (CP) CML patients (p=0.002), we hypothesized that miR-142 deficit plays a role in BC transformation. To test this hypothesis, we generated a miR-142 KO BCR-ABL (i.e., miR-142−/−BCR-ABL) mouse by crossing a miR-142−/− mouse with a miR-142+/+BCR-ABL mouse. While the miR-142+/+BCR-ABL mice developed and died of CP CML, the miR-142−/−BCR-ABL mice developed a BC-like phenotype in the absence of any other acquired gene mutations and died significantly sooner than miR-142+/+BCR-ABL CP controls (p=0.001). Leukemic stem cell (LSC)-enriched Lineage-Sca-1+c-Kit+ cells (LSKs) from diseased miR-142−/−BCR-ABL mice transplanted into congenic recipients, recapitulated the BC features thereby suggesting stable transformation of CP-LSCs into BC-LSCs in the miR-142 KO CML mouse. Single cell (sc) RNA-seq profiling showed that miR-142 deficit changed the cellular landscape of the miR-142−/−BCR-ABL LSKs compared with miR-142+/+BCR-ABL LSKs with expansion of myeloid-primed and loss of lymphoid-primed factions. Bulk RNA-seq analyses along with unbiased metabolomic profiling and functional metabolic assays demonstrated enhanced fatty acid β-oxidation (FAO) and oxidative phosphorylation (OxPhos) in miR-142−/−BCR-ABL LSKs vs miR-142+/+BCR-ABL LSKs. MiR-142 deficit enhanced FAO in miR-142−/−BCR-ABL LSKs by increasing the expression of CPT1A and CPT1B, that controls the cytosol-to-mitochondrial acyl-carnitine transport, a critical step in FAO. MiR-142 deficit also enhanced OxPhos in miR-142−/−BCR-ABL LSKs by increasing mitochondrial fusion and activity. As the homeostasis and activity of LSCs depend on higher levels of these oxidative metabolism processes, we then postulate that miR-142 deficit is a potentially druggable target for BC-LSCs. To this end, we developed a novel CpG-miR-142 mimic oligonucleotide (ODN; i.e., CpG-M-miR-142) that corrected the miR-142 deficit and alone or in combination with a tyrosine kinase inhibitor (TKI) significantly reduced LSC burden and prolonged survival of miR-142−/−BCR-ABL mice. The results from murine models were validated in BC CD34+CD38- primary blasts and patient-derived xenografts (PDXs). In conclusion, an acquired miR-142 deficit sufficed in transforming CP-LSCs into BC-LSCs, via enhancement of bioenergetic oxidative metabolism in absence of any additional gene mutations, and likely represent a novel therapeutic target in BC CML.
2023-01-13 | ST002446 | MetabolomicsWorkbench
Project description:One of the main objective of this study is to characterize Imatinib induced MSCs-mediated resistance evolution in BCR-ABL+ ALL. Tyrosine kinase inhibitor (TKI) Imatinib (IM) is used as a frontline therapy for BCR-ABL–positive (BCR-ABL+) acute lymphoblastic leukemia (ALL). However, resistance to IM therapy develops rapidly in a substantial proportion of treated patients, and the molecular mechanisms underlying the resistance are poorly understood. In this study, we identified a novel cascade of consequential events that are initiated by IM, which traverse through mesenchymal stem/stromal cells (MSCs) to leukemic cells, and lead to IM resistance. Our data showed that MSCs exposed to IM were decreased in their stemness and acquired a new functional status that enabled the formation of leukemic cell niches. These MSCs had increased expression of genes encoding chemo-attractants, adhesion molecules, and pro-survival stimulant growth factors. We found that BCR-ABL+ leukemic cells persistently exposed to IM were able to switch from BCR-ABL–driven signaling to growth factor–driven signaling for survival, and this switch was reversible. Blocking both the BCR-ABL–driven pathway and the growth factor–driven JAK pathway effectively eradicated the leukemic cell niches. Our findings illustrate TKI-induced, MSC-mediated drug resistance, suggesting an effective way to eliminate this type of drug resistance in patients with BCR-ABL+ ALL. Gene expression signatures were compared from triplicate samples of MSCs that were either treated with vehicle or imatinib for 32, 64 and 96 hours.
2015-09-01 | E-GEOD-56472 | biostudies-arrayexpress
Project description:Imatinib is highly effective in the treatment of chronic myelogenous leukemia (CML), but the primary and acquired imatinib resistance remains the big hurdle. Molecular mechanisms for CML resistance to tyrosine kinase inhibitors, beyond point mutations in BCR-ABL kinase domain, still need to be addressed. Here, we demonstrated that TXNIP is a novel BCR-ABL target gene. Suppression of TXNIP is responsible for BCR-ABL triggered glucose metabolic reprogramming and mitochondrial homeostasis. Mechanistically, Miz-1/P300 complex transactivates TXNIP through the recognition of TXNIP core promoter region, responding to the c-Myc suppression by either imatinib or BCR-ABL knockdown. TXNIP restoration sensitizes CML cells to imatinib treatment and compromises imatinib resistant CML cell survival, predominantly through the blockage of both glycolysis and glucose oxidation which results in the mitochondrial dysfunction and ATP production. In particular, TXNIP suppresses expressions of the key glycolytic enzyme, HK2 and LDHA, potentially through Fbw7-dependent c-Myc degradation. In accordance, BCR-ABL suppression of TXNIP provides a novel survival pathway for the transformation of mouse BM cells. Combination of drug inducing TXNIP expression with imatinib synergistically kills CML cells from patients and further extends the survival of CML mice. Thus, the activation of TXNIP represents an effective strategy for CML treatment to overcome resistance.
2023-05-03 | GSE218451 | GEO
Project description:One of the main objective of this study is to characterize Imatinib induced MSCs-mediated resistance evolution in BCR-ABL+ ALL. Tyrosine kinase inhibitor (TKI) Imatinib (IM) is used as a frontline therapy for BCR-ABL–positive (BCR-ABL+) acute lymphoblastic leukemia (ALL). However, resistance to IM therapy develops rapidly in a substantial proportion of treated patients, and the molecular mechanisms underlying the resistance are poorly understood. In this study, we identified a novel cascade of consequential events that are initiated by IM, which traverse through mesenchymal stem/stromal cells (MSCs) to leukemic cells, and lead to IM resistance. Our data showed that MSCs exposed to IM were decreased in their stemness and acquired a new functional status that enabled the formation of leukemic cell niches. These MSCs had increased expression of genes encoding chemo-attractants, adhesion molecules, and pro-survival stimulant growth factors. We found that BCR-ABL+ leukemic cells persistently exposed to IM were able to switch from BCR-ABL–driven signaling to growth factor–driven signaling for survival, and this switch was reversible. Blocking both the BCR-ABL–driven pathway and the growth factor–driven JAK pathway effectively eradicated the leukemic cell niches. Our findings illustrate TKI-induced, MSC-mediated drug resistance, suggesting an effective way to eliminate this type of drug resistance in patients with BCR-ABL+ ALL.
2015-09-01 | GSE56472 | GEO
Project description:Generating hematopoietic stem cells from pluripotent stem cells (PSC) has been a long lasting quest in the field of hematology. Previous studies suggested that the enforced expression of BCR-ABL, the oncogenic driver of chronic myeloid leukemia (CML), in hematopoietic cells derived from mouse embryonic stem cells (ESCs) is sufficient to confer on them in vivo long-term repopulating potential. To uncover more precisely the events regulated by BCR-ABL expression during the course of ESC differentiation, we engineered a doxycycline inducible system to modulate BCR-ABL expression.
2023-03-07 | GSE198018 | GEO
Project description:KCL-22 is a chronic myeloid leukemia (CML) cell line derived from a patient in blast crisis phase and harbors the BCR-ABL translocation. The catalytic (ATP-competitive) BCR-ABL inhibitors imatinib and nilotinib have dramatically improved CML patient outcome, but the development of resistance remains a clinical challenge. The recent identification of allosteric BCR-ABL inhibitors, such as GNF-2, which target the enzyme by binding to the myristoyl pocket rather than catalytic site of ABL1, may provide a strategy to broadly overcome resistance to the class of ABL1 ATP competitive inhibitors. We therefore wanted to use the ClonTracer barcoding system to compare the clonal responses of KCL-22 to imatinib, nilotinib and GNF-2. RNA-seq was employed to characterize genetic alterations and gene expression signatures in the pooled cell populations resistant to BCR-ABL inhibitors as well as single clones showing differential response to the three inhibitors. mRNA profiling of the subpopulations and single clones of human CML cell line KCL-22 that contribute to BCR-ABL inhibitor resistance
2014-10-07 | E-GEOD-62121 | biostudies-arrayexpress
Project description:Activating mutations of G protein alpha subunits (Ga) occur in 4-5% of all human cancers1 but oncogenic alterations in beta subunits (Gb) have not been defined. Here we demonstrate that recurrent mutations in the Gb proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Ga subunits as well as downstream effectors, and disrupt Ga-Gbg interactions. Different mutations in Gb proteins clustered to some extent based on lineage; for example, all eleven GNB1 K57 mutations were in myeloid neoplasms while 6 of 7 GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 alleles in Cdkn2a-deficient bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 mutations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling. The GM-CSF dependent human cell line TF-1 was transduced with a retrovirus expressing GNB1 K89E or empty vector. RNA was extracted from cells 12 hours after withdrawal of GM-CSF and from cells maintained in GM-CSF. Biological triplicates of each condition were performed. Gene expression differences were calculated between the four conditions.
2014-12-07 | E-GEOD-60990 | biostudies-arrayexpress
Project description:Activating mutations of G protein alpha subunits (Ga) occur in 4-5% of all human cancers1 but oncogenic alterations in beta subunits (Gb) have not been defined. Here we demonstrate that recurrent mutations in the Gb proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Ga subunits as well as downstream effectors, and disrupt Ga-Gbg interactions. Different mutations in Gb proteins clustered to some extent based on lineage; for example, all eleven GNB1 K57 mutations were in myeloid neoplasms while 6 of 7 GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 alleles in Cdkn2a-deficient bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 mutations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.
2014-12-07 | GSE60990 | GEO