Imatinib-induced Apoptosis Research Articles

Revitalizing premature ovarian failure: quercetin counteracts imatinib-induced apoptosis via the PI3K/AKT signaling pathway based on network pharmacology.

As a commonly used first-line targeted drug, imatinib (Ima) is widely used first-line treatment for cancer patients. Patient survival is significantly prolonged, but Ima can cause premature ovarian failure (POF) and affect fertility. However, the underlying mechanism is unknown, and no effective method can be employed to improve this process. To investigate the effect of quercetin (Que) on Ima-induced POF and the underlying mechanism. The therapeutic impact of Que on Ima-induced POF in mice was clarified via molecular biology experiments and in vivo experiments in animals. To verify the underlying mechanism, network pharmacology was employed to construct a signaling network of Que-Ima-POF-related genes, followed by molecular biology and docking analysis. Network pharmacology analysis identified 38 therapeutic targets of Que in Ima-induced POF. The KEGG pathways of these genes were enriched for the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Molecular docking analysis revealed that the epidermal growth factor receptor (EGFR) is a shared target of Que, Ima, and POF and has strong binding affinity. Hematoxylin-eosin (HE) staining and ELISA confirmed that Que can partially restore the ovarian index and function of mice with Ima-induced POF. Western blot, TUNEL, and immunohistochemical staining confirmed that Que promoted the PI3K/Akt signaling pathway and reduced apoptosis in Ima-induced POF mice. Thus, Que could inhibit apoptosis in Ima-induced POF by activating the PI3K/Akt pathway.

Immortalised chronic myeloid leukemia (CML) derived mesenchymal stromal cells (MSCs) line retains the immunomodulatory and chemoprotective properties of CML patient-derived MSCs

Despite the success of Tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML), leukemic stem cells (LSCs) persist, contributing to relapse and resistance. CML Mesenchymal Stromal Cells (MSCs) help in LSC maintenance and protection from TKIs. However, the limited passage and self-differentiation abilities of primary CML MSCs hinder extensive research. To overcome this, we generated and characterized an immortalised CML patient-derived MSC (iCML MSC) line and assessed its role in LSC maintenance. We also compared the immunophenotype and differentiation potential between primary CML MSCs at diagnosis, post-treatment, and with normal bone marrow MSCs. Notably, CML MSCs exhibited enhanced chondrogenic differentiation potential compared to normal MSCs. The iCML MSC line retained the trilineage differentiation potential and was genetically stable, enabling long-term investigations. Functional studies demonstrated that iCML MSCs protected CML CD34+ cells from imatinib-induced apoptosis, recapitulating the bone marrow microenvironment-mediated resistance observed in patients. iCML MSC-conditioned media enabled CML CD34+ and AML blast cells to proliferate rapidly, with no impact on healthy donor CD34+ cells. Gene expression profiling revealed dysregulated genes associated with calcium metabolism in CML CD34+ cells cocultured with iCML MSCs, providing insights into potential therapeutic targets. Further, cytokine profiling revealed that the primary CML MSC lines abundantly secreted 25 cytokines involved in immune regulation, supporting the hypothesis that CML MSCs create an immune modulatory microenvironment that promotes growth and protects against TKIs. Our study establishes the utility of iCML MSCs as a valuable model to investigate leukemic-stromal interactions and study candidate genes involved in mediating TKI resistance in CML LSCs.

FBXW7 regulates the sensitivity of imatinib in gastrointestinal stromal tumors by targeting MCL1.

Imatinib contributes to improving prognosis of high-risk or unresectable gastrointestinal stromal tumors (GISTs). As therapeutic efficacy is limited by imatinib resistance and toxicity, the exploration of predictive markers of imatinib therapeutic efficacy that enables patients to utilize more effective therapeutic strategies remains urgent. The correlation between FBXW7 and imatinib resistance via FBXW7-MCL1 axis was evaluated in vitro and in vivo experiments. The significance of FBXW7 as a predictor of imatinib treatment efficacy was examined in 140 high-risk patients with GISTs. The ability of FBXW7 to predict therapeutic efficacy of adjuvant imatinib in high-risk GIST patients was determined through 5-year recurrence-free survival (RFS) rates analysis and multivariate analysis. FBXW7 affects imatinib sensitivity by regulating apoptosis in GIST-T1 cells. FBXW7 targets MCL1 to regulate apoptosis. MCL1 involves in the regulation of imatinib sensitivity through inhibiting apoptosis in GIST-T1 cells. FBXW7 regulates imatinib sensitivity by down-regulating MCL1 to enhance imatinib-induced apoptosis in vitro. FBXW7 regulates imatinib sensitivity of GIST cells by targeting MCL1 to predict efficacy of imatinib treatment in vivo. FBXW7 regulates imatinib sensitivity by inhibiting MCL1 to enhance imatinib-induced apoptosis in GIST, and predicts efficacy of imatinib treatment in high-risk GIST patients treated with imatinib.

Abstract 956: FBXW7 regulates imatinib chemoresistance through targeting MCL1 for apoptotic regulation in gastrointestinal stromal tumors

Abstract Introduction: Imatinib definitively contribute to improve prognosis for high-risk or unresectable GISTs. Due to therapeutic efficacy is limited by imatinib chemoresistance and toxicity, the exploration of predictive markers of imatinib therapeutic efficacy that early enable patients to utilize more effective therapeutic strategies remains urgent. Here, we evaluate that the ability of FBXW7 acts as a predictor of imatinib therapeutic effect in preclinical models, and the potential of FBXW7-MCL1 axis promote imatinib-induced apoptosis response. Methods: The cellular and in vivo correlation between FBXW7 and imatinib chemoresistance via FBXW7-MCL1 axis were evaluated by detecting IC50 value, apoptosis assay, and GIST xenograft studies. The clinical significance of FBXW7 as a predictor of imatinib treatment efficacy was examined in 140 high-risk patients with GISTs by immunohistochemical staining and multivariate analyses. Results: FBXW7 affects imatinib chemoresistance through regulating cellular apoptosis in GIST-T1 cells. FBXW7 targets MCL1 to regulate cellular apoptosis. MCL1 involves in the regulation of imatinib chemoresistance through inhibiting apoptosis of GIST-T1 cells. FBXW7 regulates imatinib chemoresistance via down-regulating MCL1 for enhancing imatinib-induced apoptosis in vitro. FBXW7 regulates imatinib sensitivity of GIST cells via targeting MCL1 to predict therapeutic efficacy of imatinib treatment in vivo. Through 5-year RFS rates analysis and multivariate analysis, the ability of FBXW7 predicting therapeutic efficacy of high-risk GIST patients with adjuvant imatinib was proved. Conclusions: FBXW7 regulates imatinib chemoresistance through inhibiting MCL1 for enhancing imatinib-induced apoptosis in GIST and acted as a reliable predictor of imatinib treatment efficacy in high-risk GIST patients treated imatinib. Citation Format: Xiyu Wu, Masaaki Iwatsuki, Meiyue Lou, Takatsugu Ishimoto, Yoshifumi Baba, Yuji Miyamoto, Hideo Baba. FBXW7 regulates imatinib chemoresistance through targeting MCL1 for apoptotic regulation in gastrointestinal stromal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 956.

Effect of Cyr61 on Imatinib Resistance in Chronic Myeloid Leukemia and Its Mechanism

To investigate the effect of Cyr61 on imatinib (IM) resistance in chronic myeloid leukemia (CML) and its mechanism. Cyr61 level in cell culture supernatant was determined by enzyme-linked immunosorbent assay. The expression of Cyr61 and Bcl-xL were measured by real-time PCR and Western blot. Cell apoptosis was analyzed using an Annexin V-APC Kit. Expression of signal pathways related proteins was determined by Western blot. The level of Cyr61 obviously increased in K562G cells (IM resistance to CML cell line K562). Down-regulating the expression of Cyr61 decreased the resistance of K562G cells to IM and promoted IM induced apoptosis. In CML mouse model, down-regulating the expression of Cyr61 could increase the sensitivity of K562G cells to IM. The mechanism studies showed that Cyr61 mediated IM resistance in CML cells was related to the regulation of ERK1/2 pathways and apoptosis related molecule Bcl-xL by Cyr61. Cyr61 plays an important role in promoting IM resistance of CML cells. Targeting Cyr61 or its related effectors pathways may be one of the ways to overcome IM resistance of CML cells.

IFNγ directly counteracts imatinib-induced apoptosis of primary human CD34+ CML stem/progenitor cells potentially through the upregulation of multiple key survival factors

ABSTRACT Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon γ (IFNγ) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFNγ antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFNγ also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFNγ was independent of alternative IFNγ signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFNγ upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFNγ, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.

P694: INVESTIGATING THE ROLE OF CELL ADHESION MOLECULES IN TUNNELING NANOTUBES FORMATION IN CHRONIC MYELOID LEUKEMIA MICROENVIRONMENT

Background: The cross-talk and interactions between leukemia cells and their microenvironment play an important role in the development of drug resistance. Tunneling nanotubes (TNTs) represent a novel type of direct cell-cell communication way allowing for direct transfer of different types of cargo between distant cells. Our previous studies (Kolba et al., 2019) have shown that these structures are formed between bone marrow stromal HS-5 cells and chronic myeloid leukemia (CML) K562 cells. Significantly, TNT-mediated transfer of membrane vesicles from stromal to leukemic cells resulted in the stroma-mediated protection of CML cells from imatinib-induced apoptosis. Despite the efforts made to understand the molecular mechanisms regulating TNTs formation, they still remain unclear - especially in the leukemia microenvironment. We have shown that TNTs are formed between stromal and leukemic cells, following direct cell-cell interaction and cell dislodgement. Thus, we have focused our attention on cell adhesion molecules, as potential key players in tunneling nanotubes formation and TNT-mediated vesicles transfer between leukemic and stromal cells. Aims: The aim of presented studies was to elucidate the role of CD44 and ITGβ1 - one of the most important cell adhesion molecules, in TNTs formation between leukemic and stromal cells and TNT-mediated exchange of vesicles. Methods: To examine this, we established the HS-5 stromal cell line with silenced CD44 protein expression. Then, we co-cultured control (wt) or CD44-silenced HS-5 cells with leukemic K562 cells. For ITGβ1 studies, we used blocking anti-ITGβ1 antibody. Using confocal microscopy, three types of TNTs in co-cultures were counted: homotypic TNTs formed between stromal cells, homotypic TNTs formed between CML cells and TNTs formed between stromal and CML cells (heterotypic). Moreover, the TNT-mediated transfer of cellular vesicles from stromal to leukemic cells was assessed in these co-cultures using flow cytometry. Since CD44 expression increases in CML cells after co-culture with stroma, we examined its potential role in the drug resistance in CML acceptor cells. To do this, rhodamine-123 efflux assay was used. Results: We found that both, tunneling nanotubes number and uptake of cellular vesicles by leukemia, decreased after co-culture with CD44-silenced stromal cells and after blocking with anti-ITGβ1 antibody. Flow cytometry analyses of rhodamine-123 efflux showed significantly increased efflux pump activity in leukemic acceptor cells. Summary/Conclusion: Our data suggest that CD44 and ITGβ1 can play an important role in formation of tunneling nanotubes between leukemic and stromal cells and TNT-mediated cellular vesicles exchange. Moreover, CD44 presented in acceptor cells is involved in the development of drug resistance in leukemic cells, by promoting activity of efflux transporters.

P413: THE POTENTIAL USE OF AN ORAL HYPOMETHYLATING AGENT, OR-2100, AS A COMBINATION THERAPY FOR MYELOID MALIGNANCIES

Background: The combination therapy of hypomethylating agents (HMAs), azacytidine (AZA) and decitabine (DAC), with venetoclax (VEN), a selective BCL-2 inhibitor, have revolutionized the outcome of elderly patients with acute myeloid leukemia (AML), which have demonstrated the potential effect of HMAs as a combination therapy with molecular targeting agents for myeloid malignancies. However, there are a few studies on the combined effect of HMAs with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and the mechanism by which HMAs enhance the efficacy of molecular targeting agents remain poorly understood. We have developed OR-2100 (OR21), an oral HMA, a 5’-O-trialkylsilylated DAC. OR21 is as effective as DAC, but less myelosuppressive, in xenograft mouse models of solid and hematological malignancies (Hattori et al. Clin Epigenetics 2019, Watanabe et al. Blood 2020, Ureshino et al. Mol Cancer Ther 2021, Kamachi et al. Cancer Letters 2022). We investigated the efficacy and mechanism of OR21 in combination with VEN or TKIs for myeloid malignancies, AML, and CML. Aims: To investigate the efficacy and mechanism of the combination therapy of OR21 for myeloid malignancies. Methods: We investigated the efficacy and mechanism of OR21 in combination with VEN against AML and with TKIs against CML, in vitro and in vivo. Results: In AML, OR21 plus VEN synergistically inhibited cell growth, increased apoptosis in AML cell lines as similar to AZA and DAC, and significantly prolonged survival in a xenograft mouse model using HL60 cell line (p<0.05). Gene expression analysis using AML cell lines (HL60, KG1a) showed that OR21 plus VEN significantly downregulated VAMP7 which regulates mitophagy/autophagy to maintain mitochondrial homeostasis compared to VEN monotherapy. Consistently, OR21 plus VEN enhanced apoptosis by increasing ROS accumulation and attenuated mitophagy/autophagy response triggered by decreased mitochondrial membrane potential. The addition of mitophagy inducers, such as rapamycin and p62-mediated mitophagy inducer, to OR21 plus VEN decreased ROS accumulation and apoptosis, indicating OR21 enhanced ROS accumulation via attenuating VEN-induced mitophagy/autophagy pathway. In CML, OR21 plus TKIs (imatinib, dasatinib, ponatinib) synergistically inhibited cell growth, increased apoptosis in CML cell lines as similar to AZA and DAC, and significantly suppressed the colony-forming capacity of bone marrow CD34+ stem/progenitor cells in CML patients. OR21 plus imatinib significantly suppressed tumor growth compared to imatinib monotherapy in a xenograft mouse model using K562 cell line (p<0.05). In phosphoproteomic, transcriptome and methylome analysis (K562, KBM5), OR21 augmented imatinib-induced apoptosis by dephosphorylating of STAT and Src family proteins by upregulating several tumor suppressor genes, including PTPN6, through demethylation of their promoter CpG regions. Image:Summary/Conclusion: OR21 enhanced the anti-tumor effect in combination with the molecular agents for myeloid malignancies with different mechanisms: OR21 increased ROS accumulation in combination with VEN, and dephosphorylated BCR-ABL1 downstream protein kinases by demethylating effect in combination with TKIs. With its advantage as an oral HMA, OR21 is expected to have future clinical trials as a combination therapy for myeloid malignancies.

HucMSC exosomes promoted imatinib-induced apoptosis in K562-R cells via a miR-145a-5p/USP6/GLS1 axis

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm with increasing incidence worldwide. Growing evidence suggests that ubiquitin-specific proteases (USPs) play a role in cancer treatment. Dysregulation of miR-146a has been found in both adult and pediatric patients with acute leukemia. Knockdown of glutaminase-1 (GLS1) resulted in inhibition of tumor growth. However, the role of miR-146a-5p/USP6/GLS1 in leukemia and chemoresistance of leukemia cells remains to be elucidated. In the current study, USP6 level was increased in bone marrow aspiration specimens of patients with CML and associated with poor prognosis. USP6 was significantly upregulated in imatinib (IM)-resistant clinical samples compared with IM-sensitive samples. USP6 overexpression significantly inhibited IM-induced apoptosis of leukemia cells. Overexpressing USP6 significantly increased GLS1 ubiquitination to decrease GLS protein. A mechanism study indicated that USP6 regulation of IM resistance of CML cells was GLS1 dependent and regulated by miR-146a-5p. Administration of human umbilical cord mesenchymal stem cell (hucMSC) exosomes promoted IM-induced cell apoptosis through miR-145a-5p/USP6. Therefore, hucMSC exosomes promoted IM-induced apoptosis of K562-R cells by suppressing GLS1 ubiquitination to increase GLS protein via miR-146a-5p and its target GLS1. The findings highlight the importance of miR-146a-5p/USP6/GLS1 signaling in chemoresistance of leukemia and provide new insights into therapeutic strategies for chemoresistant leukemia.

Aberrantly reduced expression of miR-342-5p contributes to CCND1-associated chronic myeloid leukemia progression and imatinib resistance

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome, and the current standard of care is the use of tyrosine kinase inhibitors (TKI). However, some patients will not achieve a molecular response and may progress to blast crisis, and the underlying mechanisms remain to be clarified. In this study, next-generation sequencing was used to explore endogenous miRNAs in CML patients versus healthy volunteers, and miR-342-5p was identified as the primary target. We found that miR-342-5p was downregulated in CML patients and had a significant inhibitory effect on cell proliferation in CML. Through a luciferase reporter system, miR-342-5p was reported to target the 3’-UTR domain of CCND1 and downregulated its expression. Furthermore, overexpression of miR-342-5p enhanced imatinib-induced DNA double-strand breaks and apoptosis. Finally, by analyzing clinical databases, we further confirmed that miR-342-5p was associated with predicted molecular responses in CML patients. In conclusion, we found that both in vivo and in vitro experiments and database cohorts showed that miR-342-5p plays a key role in CML patients, indicating that miR-342-5p may be a potential target for future CML treatment or prognostic evaluation.

Induction of autophagy has protective roles in imatinib-induced cardiotoxicity.

Cardiotoxicity is one of the severe adverse effects of chemotherapeutic agents. Imatinib was previously reported to induce cardiotoxicity. Autophagy is an intracellular bulk protein and organelle degradation process, but its roles in cardiac diseases are unclear. We examined whether imatinib induces cardiomyocyte autophagy, and the role of autophagy in imatinib-induced cardiotoxicity using in vitro and in vivo experiments. In in vitro experiments, neonatal rat cardiomyocytes were treated with imatinib (1, 5, or 10 μM; 6 h). Myocyte autophagy was assessed by microtubule-associated protein light chain (LC) 3-II, beclin 1, mature cathepsin D, and acridine orange-stained mature autolysosome expression. Imatinib increased their expression, suggesting that it induced autophagy. Consequently, imatinib altered the production of mitochondria-derived reactive oxygen species (ROS) and loss of mitochondrial membrane potential, which were assessed by the fluorescent indicator MitoSOX and JC-1, respectively, leading to cardiomyocyte apoptosis. 3-methyl-adenine (3MA), an autophagic inhibitor, exacerbated imatinib-induced apoptosis by 30 %. In in vivo experiments, C57BL/6 mice were treated with imatinib (50 and 200 mg/kg/day) for 5 weeks in the presence or absence of 3MA. Echocardiographic measurement revealed that imatinib (200 mg) caused dilatation of the left ventricle (LV) and reduced LV fractional shortening. Apoptosis and LC3-II expression in cardiac tissue were increased by imatinib. Co-treatment with 3MA and imatinib further impaired imatinib-induced cardiac apoptosis and LV dysfunction. This study suggests that imatinib induces cardiomyocyte apoptosis, leading to cardiac dysfunction. Imatinib increases cardiomyocyte autophagy as a consequence of apoptosis and autophagy has a pro-survival role in imatinib-induced cardiac impairment.

KDM6A promotes imatinib resistance through YY1-mediated transcriptional upregulation of TRKA independently of its demethylase activity in chronic myelogenous leukemia

Rationale: Despite landmark therapy of chronic myelogenous leukemia (CML) with tyrosine kinase inhibitors (TKIs), drug resistance remains problematic. Cancer pathogenesis involves epigenetic dysregulation and in particular, histone lysine demethylases (KDMs) have been implicated in TKI resistance. We sought to identify KDMs with altered expression in CML and define their contribution to imatinib resistance.Methods: Bioinformatics screening compared KDM expression in CML versus normal bone marrow with shRNA knockdown and flow cytometry used to measure effects on imatinib-induced apoptosis in K562 cells. Transcriptomic analyses were performed against KDM6A CRISPR knockout/shRNA knockdown K562 cells along with gene rescue experiments using wildtype and mutant demethylase-dead KDM6A constructs. Co-immunoprecipitation, luciferase reporter and ChIP were employed to elucidate mechanisms of KDM6A-dependent resistance.Results: Amongst five KDMs upregulated in CML, only KDM6A depletion sensitized CML cells to imatinib-induced apoptosis. Re-introduction of demethylase-dead KDM6A as well as wild-type KDM6A restored imatinib resistance. RNA-seq identified NTRK1 gene downregulation after depletion of KDM6A. Moreover, NTRK1 expression positively correlated with KDM6A in a subset of clinical CML samples and KDM6A knockdown in fresh CML isolates decreased NTRK1 encoded protein (TRKA) expression. Mechanistically, KDM6A was recruited to the NTRK1 promoter by the transcription factor YY1 with subsequent TRKA upregulation activating down-stream survival pathways to invoke imatinib resistance.Conclusion: Contrary to its reported role as a tumor suppressor and independent of its demethylase function, KDM6A promotes imatinib-resistance in CML cells. The identification of the KDM6A/YY1/TRKA axis as a novel imatinib-resistance mechanism represents an unexplored avenue to overcome TKI resistance in CML.

Abstract 3767: FBXO32 ubiquitin ligase mediates apoptosis evasion and adaptation to KIT inhibition in gastrointestinal stromal tumor - GIST

Abstract Introduction: GIST is the most common malignant mesenchymal neoplasm and features myogenic differentiation. Most GISTs depend upon oncogenic signaling through constitutively activated forms of KIT and exhibit remarkable responses to the first-line KIT inhibitor imatinib. Secondary resistance involves emergence of subclonal populations harboring heterogeneous KIT-secondary mutations that are not collectively inhibited by any approved therapy. We dissected KIT-downstream signaling nodes to identify mediators of the KIT oncogenic program, seeking targets of overarching relevance in polyclonal imatinib-resistant GIST. Methods: using clinically-representative human GIST cell models, we undertook pharmacological and functional screening to identify key targetable nodes within KIT-downstream PI3K/mTOR and RAS/MAPK pathways and to model therapeutic strategies in vitro and in vivo. Transcriptomic analysis (RNAseq) identified genes co-regulated by PI3K/mTOR and RAS/MAPK, and functional evaluations by lentiviral construct overexpression and gene knockdown elucidated roles in GIST biology. Results: PI3K/mTOR and MEK1/2 were the most essential KIT-dependent nodes irrespective of the type of KIT primary or secondary mutation. Single node ablation did not yield sustained antiproliferative and apoptotic effects, but concurrent intermittent inhibition was synergistic in vitro and in vivo, supporting the critical and complementary roles of these two pathways. Transcriptomic analyses underscored FBXO32 (a SCF E3 ubiquitin ligase and the main effector of muscular atrophy) as the most differentially expressed gene co-regulated by the PI3K/mTOR and RAS/MAPK pathways. FBXO32 was also the most differentially expressed gene after KIT inhibition in GIST cell lines, and enrichment in FBXO32 expression was found in post-imatinib GIST samples compared to baseline matched tumor tissue. We demonstrated that FBXO32 is transcriptionally upregulated by FOXO3a upon KIT or concurrent PI3K/mTOR and MEK1/2 blockage, paralleling human muscle regulation. Notoriously, FBXO32 proved to have a pro-survival role in GIST cells, enabling evasion of apoptosis. Microarray and in vitro and in vivo functional assays showed that FBXO32 mediates cell cycle exit and quiescence upon KIT or concurrent KIT-downstream pathways inhibition, thereby minimizing imatinib-induced apoptosis and facilitating GIST cell survival. Conclusions: PI3K/mTOR and MEK1/2 are critical mediators of KIT oncogenic signaling in GIST. FBXO32 emerges as a KIT-dependent E3 ubiquitin ligase transcriptionally repressed through KIT downstream pathways. However, FBXO32 induction upon therapeutic KIT signaling inhibition triggers quiescence and subsequent apoptosis evasion, thus emerging as a novel mechanism of adaptation to therapeutic inhibition of oncogenic KIT. Citation Format: Alfonso García Valverde, Jordi Rosell, Daniel Pilco Janeta, Sergi Sayols, Claudia Valverde, Anna Esteve, Marta Gut, Jordi Barretina, George D. Demetri, Jonathan A. Fletcher, Joan Carles, Joaquín Arribas, César Serrano. FBXO32 ubiquitin ligase mediates apoptosis evasion and adaptation to KIT inhibition in gastrointestinal stromal tumor - GIST [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3767.

Abstract 648: Role of G0S2 in chronic myeloid leukemia and TKI resistance

Abstract Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic disease. However, resistance is a clinical problem, and TKIs do not target CML leukemic stem cells (LSC), which are independent from BCR-ABL1 kinase activity. To understand mechanisms driving BCR-ABL1-independent resistance, we analyzed the transcriptional signature of TKI-naïve CD34+ cells from patients with or without a response to imatinib after 12 months of therapy (McWeeney et al. Blood 2010). Among the genes most profoundly downregulated in patients destined to fail imatinib was G0/G1 switch gene 2 (G0S2) (&amp;gt;3-fold, n=59, p&amp;lt;0.02). Retrospective analysis of survival data for patients whose samples were evaluated in a microarray revealed that high G0S2 expression (&amp;gt;50%) correlated with a longer overall survival (n=30 responders, n=16 non-responders, p=0.036). We next analyzed G0S2 mRNA expression in CD34+ cells from normal cord blood and from primary CD34+ cells lacking BCR-ABL1 kinase domain mutations. G0S2 was 4-fold downregulated in newly diagnosed CML (n=6) compared to normal cord blood (n=5, p&amp;lt;0.01), and further downregulated by &amp;gt;3-fold in TKI-resistant (n=2) and BP-CML samples (n=5, p&amp;lt;0.01).G0S2 mRNA was also lower in CD34+38- stem cells compared to committed CD34+38+ progenitor cells in CML (n=6, p&amp;lt;0.01) but not normal cord blood (n=4, p=ns). CFSE staining revealed that G0S2 mRNA levels in CP-CML CD34+ cells are lowest in cell populations with the least number of cell divisions. To assess the role of G0S2 in CML, we cloned G0S2 amplified from normal human mononuclear cells into a lentiviral expression system, and confirmed ectopic expression by qRT-PCR and immunoblot. Ectopic G0S2 reduced colony formation in both TKI-sensitive and TKI-resistant CML cell lines and CD34+ CML patient samples. While G0S2 has been reported to induce apoptosis by direct inhibition of BCL-2, reduced colony formation was not associated with an increase of apoptosis, but did restore imatinib-induced apoptosis. These data are consistent with a tumor suppressor role for G0S2 in CML. G0S2 has also been shown to inhibit adipocyte triglyceride lipase (ATGL), the rate-limiting enzyme in the conversion of triglycerides to free fatty acids (FFAs). While ATGL is indeed expressed at the protein level in CML cells, shRNAs targeting ATGL do not phenocopy ectopic G0S2 expression. Furthermore, the effects of ectopic G0S2 are not impaired in cells with simultaneous ATGL knockdown, and ectopic G0S2 has no effect on intracellular FFA or ATP levels. Finally, G0S2 gene expression has been shown to be regulated by promoter DNA hypermethylation. However, DNA bisulfite and patch PCR sequencing in primary CD34+ cells from CML patients did not reveal G0S2 promoter hypermethylation in CML. Rather, ChIP-PCR revealed the presence of MYC/MAX at the G0S2 promoter in CML, suggesting MYC-mediated transcriptional repression. Altogether, these data suggest that G0S2 downregulation plays a functional role in CML disease progression and TKI response, but is independent from its role as an inhibitor of BCL-2 or ATGL. These data unravel a new role for G0S2 as a regulator of TKI response in CML, and suggest that restoring G0S2 expression may have clinical utility. Citation Format: Mayra A. Gonzalez, Alfonso E. Bencomo, Christian Barreto-Vargas, Andres J. Rubio, Idaly M. Olivas, Joshua J. Lara, Anna Senina, Jonathan Ahmann, Katherine T. Varley, Luis F. Jave-Suarez, O'Hare Thomas, Michael W. Deininger, Anna M. Eiring. Role of G0S2 in chronic myeloid leukemia and TKI resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 648.

P6275Role of autophagy in imatinib-induced cardiotoxicity

Abstract Background Cardiotoxicity is one of the severe adverse effects of chemotherapeutic agents. Imatinib, a therapeutic agent for chronic myelogenous leukemia, has been reported to induce cardiotoxicity. Autophagy is an intracellular bulk protein and organelle degradation process, but it is unclear whether autophagy functions as pro-death or pro-survival program during disease conditions. We examined whether imatinib induces myocyte autophagy and the role of autophagy in imatinib-induced cardiotoxicity in in vitro and in vivo experiments. Methods In in vitro experiments, neonatal rat cardiac myocytes were treated with imatinib (1, 5, 10 μM; 1–6 hrs). Inhibition of autophagy was performed using 3-methyl-adenine (3MA), an autophagic inhibitor, and transfection with Atg5-targeted siRNA. Myocyte apoptosis was detected by morphological change in nuclei and caspase 3 activity. Mitochondria-derived reactive oxygen species production was detected using MitoSOX and mitochondrial membrane potential was assessed by TMRM staining. Expressions of cytochrome c in mitochondria and cytosole were examined by Werstern blotting. Myocyte autophagy was assessed by monodansylcadaverine staining and microtubule-associated protein light chain (LC) 3-II expression. In in vivo experiments, C57BL6 mice were treated with imatinib (50 and 200 mg/kg/day) for 5 weeks in the presence or absence of 3MA. Cardiac function was examined by echocardiography. In cardiac tissue, apoptotic myocytes were examined by TUNEL assay and autophagy was examined by LC3-II expression. Results In in vitro experiments, imatinib increased apoptotic nuclei and caspase 3 activity, in a dose-dependent manner. Consequently, imatinib augmented production of mitochondria-derived reactive oxygen species, loss of mitochondrial membrane potential, and the release of cytochrome c from mitochondria to cytosole, suggesting that imatinib induced mitochondrial-apoptotic pathway. On the other hand, imatinib significantly increased monodansylcadaverine stained dots and LC3-II expression, suggesting that imatinib increased autophagy. 3MA and Atg5 siRNA augmented imatinib-induced apoptosis by 60% and 30%, respectively. In in vivo experiments, imatinib (200 mg) exhibited the dilatation of left ventricle by 15% and the depression of left ventricular fractional shortening by 23%. Ratio of apoptotic myocytes was significantly increased and LC3-II expression in cardiac tissue was enhanced by imatinib in a dose-dependent fashion. Co-treatment with 3MA and imatinib further impaired imatinib-induced myocyte apoptosis by 3 fold and LV dysfunction by 20%. Conclusion These results indicate that imatinib induced myocyte apoptosis, leading to cardiac dysfunction. Imatinib enhanced myocyte autophagy as a consequence of apoptosis and autophagy was a beneficial phenomenon in this condition.

TAL1 mediates imatinib-induced CML cell apoptosis via the PTEN/PI3K/AKT pathway

Chronic myeloid leukemia (CML) is associated with chromosomal translocation t(9; 22), which results in formation of the BCR-ABL oncogene. CML is treated with tyrosine kinase inhibitors (TKIs), which target BCR-ABL, to eradicate BCR-ABL + cells. However, the TKI imatinib (IM) fails to eliminate quiescent leukemia stem cells (LSCs) in CML. In this study, we demonstrate that transcription factor TAL1 is down-regulated in CML LSCs by BCR-ABL, and IM triggers TAL1 mRNA expression. In addition, loss of TAL1 abrogates IM-induced CML cell apoptosis. RNA-seq analysis suggests that TAL1 expression may affect PI3K/AKT pathway. Moreover, depletion of TAL1 inhibits the expression of PTEN, which is a negative regulator of the PI3K/AKT pathway. Our results reveal an unexpected involvement of TAL1 in CML etiology and demonstrate that TAL1 may regulate PTEN expression and lead to inhibition of the PI3K/AKT pathway in the response of CML cells to TKI. These results implicate regulation of PTEN expression as a novel mechanism for the transcriptional regulatory networks of TAL1 in CML.

Hyaluronan abrogates imatinib-induced senescence in chronic myeloid leukemia cell lines

Hyaluronan (HA) is the main glycosaminoglycan of the extracellular matrix. CD44 is the most important HA receptor, and both have been associated with poor prognosis in cancer. Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively activated tyrosine kinase (Breakpoint Cluster Region - Abelson murine leukemia viral oncogene homolog1, BCR-ABL). It is mainly treated with BCR-ABL inhibitors, such as imatinib. However, the selection of resistant cells leads to treatment failure. The aim of this work was to determine the capacity of HA (high molecular weight) to counteract the effect of imatinib in human CML cell lines (K562 and Kv562). We demonstrated that imatinib decreased HA levels and the surface expression of CD44 in both cell lines. Furthermore, HA abrogated the anti-proliferative and pro-senescent effect of Imatinib without modifying the imatinib-induced apoptosis. Moreover, the inhibition of HA synthesis with 4-methylumbelliferone enhanced the anti-proliferative effect of imatinib. These results suggest that Imatinib-induced senescence would depend on the reduction in HA levels, describing, for the first time, the role of HA in the development of resistance to imatinib. These findings show that low levels of HA are crucial for an effective therapy with imatinib in CML.

Novel AF1q/MLLT11 favorably affects imatinib resistance and cell survival in chronic myeloid leukemia

Tyrosine kinase inhibitor treatment of chronic myeloid leukemia (CML) has demonstrated beneficial effects. However, resistance to tyrosine kinase inhibitors and disease relapse are still a challenge for CML therapy. In this study, we analyzed bone marrow samples from 149 CML patients and 15 control donors, and investigated the affect of AF1q on CML cell survival and engraftment in vitro and in vivo. We found that AF1q/MLLT11 expression was significantly upregulated in CML patients, especially in CD34+ CML cells. Elevated AF1q expression was associated with disease progression. Knockdown of AF1q enhanced imatinib sensitivity, induced apoptosis, and suppressed growth in CML cells. Moreover, AF1q deficiency sensitized CD34+ CML cells to imatinib. In contrast, upregulation of AF1q promoted cell survival, protected CML cells from imatinib-induced apoptosis, and increased engraftment of CML cells in vivo. We further identified a positive correlation between AF1q and CD44 expression in chronic phase CML patients and CD34+ CML cells. Importantly, AF1q contributes to imatinib-resistance in CML by regulating the expression of CD44. These findings reveal a novel BCR-ABL-independent pathway, AF1q/CD44, involves imatinib resistance in CML, thus representing a potential therapeutic target for imatinib-resistant CML patients.

Costunolide promotes imatinib-induced apoptosis in chronic myeloid leukemia cells via the Bcr/Abl-Stat5 pathway.

Costunolide, a sesquiterpene lactone, is a small molecular monomer extracted from Inula helenium (Compositae). In the present study, we assessed the antileukemia effects of costunolide on the human chronic myeloid leukemia cell line K562 and its combined activity with imatinib. A Cell Counting Kit-8 assay demonstrated that costunolide significantly inhibited K562 cell proliferation and enhanced imatinib-induced anti-proliferative activity. We found that costunolide significantly induced mitochondrial apoptosis in K562 cells by modulating the protein levels of Bcl-2 family members and by inducing caspase activation. Costunolide promoted imatinib-induced apoptosis via the Bcr/Abl-signal transducer and activator of transcription 5 pathway. Costunolide inhibited proliferation by inducing cell cycle arrest in the G2 /M phase by decreasing cyclin B1 and cyclin-dependent kinase 2 expression and increasing p21 expression. Together, these results demonstrate that costunolide may be a potent therapeutic agent against chronic myeloid leukemia.

CD69 partially inhibits apoptosis and erythroid differentiation via CD24, and their knockdown increase imatinib sensitivity in BCR-ABL-positive cells.

Chronic myeloid leukemia (CML) is caused by a constitutively active BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKIs) imatinib and its derivatives represent a breakthrough for CML therapy, but the use of TKI alone is ineffective for many CML patients. CD69, an early activation marker of lymphocytes, participates in immune and inflammatory responses. Previous studies revealed that BCR-ABL upregulates CD69 expression; however, the role of CD69 in CML cells is unknown. Here, we demonstrate that BCR-ABL induced CD69 promoter activity and mRNA and protein expression via the NF-κB pathway. CD69 knockdown partially increased apoptosis and expression of erythroid differentiation markers, α-globin, ζ-globin, and glycophorin A, and increased imatinib sensitivity in K562 and KU812 CML cells. Gene microarray analysis and quantitative real-time PCR verified that CD24, an oncogenic gene, downregulated in K562 cells upon CD69 knockdown. CD69 overexpression increased, whereas CD69 knockdown inhibited CD24 promoter activity and mRNA and protein levels. CD24 knockdown also partially increased apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells, whereas its overexpression inhibited the effects of CD69 knockdown on apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells. Imatinib-induced apoptosis and erythroid differentiation were also inhibited by CD69 or CD24 overexpression in BCR-ABL-expressing CML cell lines and CD34+ cells. Taken together, CD24 is a downstream effector of CD69. CD69 and CD24 partially inhibit apoptosis and erythroid differentiation in CML cells; thus, they may be potential targets to increase imatinib sensitivity.