CML Leukemia Stem Cells Research Articles

Clinical Insights into Structure, Regulation, and Targeting of ABL Kinases in Human Leukemia.

Chronic myeloid leukemia is a multistep, multi-lineage myeloproliferative disease that originates from a translocation event between chromosome 9 and chromosome 22 within the hematopoietic stem cell compartment. The resultant fusion protein BCR::ABL1 is a constitutively active tyrosine kinase that can phosphorylate multiple downstream signaling molecules to promote cellular survival and inhibit apoptosis. Currently, tyrosine kinase inhibitors (TKIs), which impair ABL1 kinase activity by preventing ATP entry, are widely used as a successful therapeutic in CML treatment. However, disease relapses and the emergence of resistant clones have become a critical issue for CML therapeutics. Two main reasons behind the persisting obstacles to treatment are the acquired mutations in the ABL1 kinase domain and the presence of quiescent CML leukemia stem cells (LSCs) in the bone marrow, both of which can confer resistance to TKI therapy. In this article, we systemically review the structural and molecular properties of the critical domains of BCR::ABL1 and how understanding the essential role of BCR::ABL1 kinase activity has provided a solid foundation for the successful development of molecularly targeted therapy in CML. Comparison of responses and resistance to multiple BCR::ABL1 TKIs in clinical studies and current combination treatment strategies are also extensively discussed in this article.

Wnt and Notch Pathways Regulate Stemness of Myeloid Leukemia Cells Via IL33/ST2 Signaling

Therapy-resistant leukemic stem cells (LSCs) are a main cause of relapse in chronic and acute myeloid leukemia (CML and AML) patients. Stemness, a cellular state of LSCs characterized by their quiescence, pluripotency, and long-term self-renewal capacity, is thought to have major implications on therapy resistance and outcome in myeloid leukemias. However, signaling pathways that induce stemness are often not specific for LSCs and shared with normal hematopoietic stem cells (HSCs). Therefore, LSCs are difficult to be targeted therapeutically. IL-33/ST2 signaling may be a potential target to eradicate CML and AML LSCs. To investigate the ST2 gene (IL1RL1) expression in different hematological malignancies, we used the MILE gene expression dataset containing 2096 samples. The IL1RL1 high expressing samples (n=62) were exclusively represented by myeloid leukemias (CML and AML), and 50% of the IL1RL1 high samples harbored either the BCR/ABL1 (CML) or the AML1/ETO (AML) oncogenes. We confirmed the result from the MILE database by analyzing the ST2 protein expression on FACS-sorted CD34+ LSCs from AML and CML patient samples. Indeed, AML1/ETO and BCR/ABL1 harboring LSCs expressed ST2, while healthy hematopoietic stem cells (HSCs) and LSCs harboring other oncogenes were ST2-negative. Transfecting the ST2-negative cell line Ramos with the AML1/ETO and BCR/ABL1 oncogenes induced ST2-expression. Next, we performed a gene set enrichment analysis (GSEA), comparing the IL1RL1 high vs. IL1RL1 low samples in the MILE dataset. Genes involved in stemness (p<0.01), Wnt- (p<0.001), and NF-ΚB (p=0.054) signaling were upregulated, while the Notch-pathway (p<0.05) was downregulated. Since the NF-ΚB is a known downstream pathway of ST2, we hypothesized that Wnt and/or Notch signaling pathways regulate ST2 expression. Therefore, we treated FACS-sorted CD34+ AML1/ETO expressing LSCs with the Wnt pathway inhibitor XAV939 or HES1 and NOTCH1 shRNAs, silencing the Notch pathway. Treatment with XAV939 led to a decrease in ST2 expression in these cells, while silencing HES1 and NOTCH1 enhanced ST2-expression. To analyze the molecular mechanisms induced by IL-33/ST2 signaling, we performed a transcriptomic analysis using FACS-sorted CD34+ AML1/ETO LSCs upon treatment with or without IL-33. In total, 377 genes were up- and 139 genes downregulated after IL-33 stimulation. IL-33-induced genes were involved in the positive regulation of NF-ΚB (p<0.05) and canonical Wnt (p<0.001) signaling pathways, and the regulation of stem cell division and maintenance (p<0.001). To functionally test whether IL-33/ST2 signaling triggers stemness in myeloid leukemia, we performed a colony forming assay using CD34+ AML1/ETO or BCR/ABL1 expressing LSCs from leukemia patient samples. The colony numbers increased 1.5-3fold upon IL-33 treatment in three consecutive platings, indicating an increase in stem cell numbers. IL-33 had no effect on colony numbers of HSCs and LSCs harboring other oncogenes. To test the in vivo relevance of IL-33/ST2 signaling in leukemia development, we used a congenic AML1/ETO9a (AE9a) or BCR/ABL1 (CML) mouse model. Briefly, we transduced ST2-negative HSCs originating from BL/6 or ST2-knockout (ST2-/-) mice with AE9a-GFP or BCR/ABL1-GFP and transplanted those cells into recipients. In both models, the LSCs strongly expressed ST2, but IL-33/ST2 signaling had no influence on the disease development. Stemness supports resistance against standard therapy in both, AML and CML. Since we were able to show that IL-33/ST2 signaling promotes stemness in AML1/ETO and BCR/ABL1 LSCs, we hypothesized that IL-33 addition protects LSCs from treatment with standard therapeutics. Indeed, IL-33 rescued the reduced colony forming capacity of LSCs that were either treated with the chemotherapeutic agent cytarabine (AML1/ETO LSCs) or the tyrosine kinase inhibitor Nilotinib (BCR/ABL1 LSCs). Furthermore, ST2-deficiency led to a reduced LSC count in CML mice compared to control mice when treated with Nilotinib (p=0.047). We showed that ST2 is expressed on AML1/ETO and BCR/ABL1 harboring LSCs and that activated IL-33/ST2 signaling induced stemness in these LSCs. Targeting IL-33/ST2 signaling in these specific subtypes of AML and CML may eliminate LSCs and overcome resistance towards chemotherapy or TKIs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Real World Outcomes of Chronic Myeloid Leukemia in Era of Tyrosine Kinase Inhibitors a Single Center Experience

Introduction: Obtaining a major molecular response (MMR) correlates with a higher probability of achieving a complete molecular response (CMR) and suboptimal molecular responses are associated with increased risk of resistance. Methods: We retrospectively analyzed patients with a diagnosis of chronic myeloid leukemia who were treated at Penn State Cancer Institute between January 2008 to December 2018. These patients had bone marrow biopsy for confirmation for diagnosis of CML and were monitored by bcr-abl PCR to document molecular response: Complete (CMR ) vs. Major (MMR). Results: A total of 74 patients F :32; M:42 were diagnosed with CML between 2008 and 2018 with median age at diagnosis 52 years (22yrs -89yrs). The majority of these patients had CML in chronic phase (n=68;92% ) and 4%(n=3) were in accelerated phase (CML AP) and 4% (n=3) had blastic phase (CML BC). High Sokal score was seen in 42% (31/74) patients with concordance seen in most patients with high Sokal scores.Highest Sokal score was infinity in a patient with massively enlarged spleen with Hasford score 2179. Highest Hasford score was 5625 with discordant Sokal score of 558. Imatinib was used in front line in 45 /74 patients. Dasatinib and nilotinib were used as first line 18 vs 5, second line in 19 vs 6 and third line in 3 vs 7 patients. Ponatinib was used second line in 2 patients vs. third, forth and fifth line in 1 patient each. A total of 8 patients received allogeneic transplant ( 1 cord ) and all 8/8 achieved CMR remained in CR for medan of 12 years . Only 30/66(45%) achieved CMR who did not undergo transplant and 24/66 (36%) achieved MMR and 10 (15%) patients did not achieve MMR and remaining had progression. TKI resistance and TKI intolerance was seen in 7/66 (4.6%) patients. second cancers were seen in 9% (n=6) patients. Conclusion: Allogeneic transplant still appears to have role to achieve long term CR. Use of drugs targeting CML leukemia stem cells along with TKIs may be needed to yield more complete metabolic responses (CMR) cure CML.

CML-313 Kinetics of CITED2 Gene Expression in Chronic Myeloid Leukemia Patients

In CML, BCR-ABL1 oncoprotein induces overexpression of STAT5 and its target genes for example CITED2 gene. This gene is involved in regulating the proliferation and quiescence states of leukemic stem cells (LSCs). Recently, studies reported that adding pioglitazone (PPARγ agonist approved for T2DM treatment) to imatinib may decrease the transcription of both STAT5 and CITED2 gene, eliminate the quiescent LSCs (resist TKIs), and ameliorate the patients' response. The primary objective is to clarify the value of combining pioglitazone with imatinib to downregulate the CITED2 gene aiming to eradicate the LSCs in CML treatment. In the context of a clinical trial (approved by the Ethical Committee of the Faculty of Medicine Mansoura University), the study group patients (N=26) were treated with combination therapy after having their consent. The planned follow-up time is 2 years. Hematology unit, Oncology Center, Mansoura University. The eligibility criteria for study group patients were de novo, Philadelphia+ve CML (chronic phase) aged 18-60 years old. Those patients were treated with imatinib 400 mg and pioglitazone 15 mg once daily for 6 months. Pre- and post-treatment samples (after 6 months) were collected to assess the expression levels of the CITED2 gene. Responders continued on the same TKI and their sequential BCR/ABL Q-PCR was monitored. The study proposed that this combination therapy can put more patients into deeper and faster molecular response via eliminating the LSCs. The study group included 15 males (57.7%) and 11 females (42.3%) with a mean age of 43.2 years. The BCR-ABL and CITED2 genes' pretreatment expression levels decreased significantly after 6 months of combined treatment (p<0.001 and p=0.005, respectively). Moreover, at 6 months 65.4% and 23% of the patients had response ≥ CCyR and ≥ MMR respectively. The most frequent complaints among patients were increased body weight and edema (p<0.001). Adding pioglitazone to imatinib improved the patients' response and downregulate the overexpressed CITED2 gene. A longer follow-up is planned and needed to confirm the safety and efficacy of the combination.

Abstract 3153: Imatinib mesylate treatment improves reprogramming efficiency, and morphology of chronic myeloid leukemia derived induced pluripotent stem cells

Abstract Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL1 fusion gene with constitutively active tyrosine kinase activity. Although the tyrosine kinase inhibitors (TKI) have revolutionized the treatment for CML, withdrawal of TKI therapy in patients on deep molecular response causes disease relapse, primarily due to the persistence of leukemic stem cells (LSCs) that are insensitive to TKI. As LSCs are a rare population of cells, it is challenging to understand the molecular basis of the disease relapse to tailor strategies to eliminate them selectively. Induced pluripotent stem cells (iPSCs) derived from primary CML are increasingly used for disease modeling and high through drug screening. Cryopreserved CD34+ from three chronic phase CML patients (n=3) and CD34+ cells from a normal donor after mobilization were expanded in SFEM II supplemented with CD34+ Expansion Supplement (10X) including UM729 for 3-days and were nucleofected with episomal reprogramming plasmids as described previously (Manian et al., 2018). The nucleofection efficiency was 15% in the expanded CD34+ CML and normal cells. While the normal CD34+ cells formed iPSCs with the characteristic flat morphology between 20-24 days, the CML CD34+ cells formed several dome-shaped colonies after 30 days. Two normal CD34+ iPSC colonies continued to maintain their morphology for 12 passages. All the CML iPSC colonies (n=11) expressed the same type of BCR-ABL1 transcript as the CD34+ cells and did not carry any mutations in the BCR-ABL1 kinase domain. However, only three of the 11 CML-iPSC colonies could be maintained without significant differentiation after five passages. They lacked typical iPSC morphology and appeared as small cell aggregates or dome-shaped colonies. To understand whether this atypical morphology of the CML iPSC colonies was due to the expression of the BCR-ABL fusion protein within the cells, the medium was supplemented with 10μM imatinib. There was an increase in cell proliferation rate and gain of typical iPSCs morphology similar to normal iPSCs within two days after adding IM. Withdrawal of IM from the IM-treated CML iPSC resulted in a dome-shaped morphology, reduced proliferation rate, and reduction in TRA1-60 expression after 96 hrs. The CML iPSC cultured in the presence of IM showed significant downregulation of phospho-CRKL, a BCR-ABL1 downstream signaling protein, compared to the colonies cultured in the absence of IM. These results suggest that suppression of BCR-ABL1 by treatment with IM produced stable CML iPSC-like phenotype while IM withdrawal resulted in dome-shaped colonies and pushed the cells towards differentiation. This proof-of-concept study identified a unique CML LSCs mimetic model. It would serve as an excellent platform for screening small molecules to eliminate LSCs as it thrives on TKI therapy. Citation Format: Esther Benjamin, Gaurav Joshi, Bharathi Rajamani, Krittika Nandy, Shaji Velayudhan, Poonkuzhali Balasubramanian. Imatinib mesylate treatment improves reprogramming efficiency, and morphology of chronic myeloid leukemia derived induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3153.

Abstract 3117: CD80 can be the marker of chronic myeloid leukemia stem cells and regulated by BCR-ABL signaling

Abstract Chronic myeloid leukemia (CML) is a hematological malignancy caused by an abnormal tyrosine kinase, BCR-ABL1. Leukemic stem cells (LSCs) in this disease proliferate indefinitely. Also, the LSCs normally differentiate into mature neutrophils. Although tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL1 improved prognosis of CML patients, many patients still have residual diseases and need life-long treatment with the TKIs. This is due to a small number of CML LSCs that cannot be completely eradicated by the TKIs. It has been hypothesized that not only chemotherapy alone, but also immune surveillance systems of patients are needed to completely eradicate cancerous cells from their body. We hypothesized that residual CML cells might escape from the immune surveillance. Surfaces molecules associated with immune surveillance are examined on CML cells. At first, we confirmed expression levels of CD80 and PD-L1 by flowcytometry using CML cell lines. Our data showed that CD80 was expressed on surfaces of CML derived cell, K562, not PD-L1. Moreover, an engineered K562 subline is generated with the constitutive active JAK2V617F gene and became dependent on JAK-STAT signaling, but not BCR-ABL. This subline did not show CD80 on the surface. The TKIs inhibited expression of CD80 on the parental K562. Meanwhile, an ERK inhibitor decreased CD80 expression in K562. These results suggested that CD80 was downstream of BCR-ABL-ERK signaling pathway, not JAK-STAT pathway. When K562 cells were differentiated into mature neutrophils by ATRA, levels of CD80 decreased. Cytotoxicity assays using K562 and NK cells from normal individuals showed that the parental K562 cells and K562 cells with decreased levels of CD80 were similarly killed. These data suggested that CD80 on K562 played a minimum role in NK cell immune surveillance. We also found that only a small fraction of CD11b positive monocytes in normal peripheral blood showed expression of CD80. Also, blastic cells of CML blastic phase showed CD80 expression. These results suggested that only blastic cells of CML and a fraction of monocytes expressed CD80. These cells were distinguished by expression of CD11b as a marker. Although it is currently impossible to distinguish normal stem cells from CML LSCs, our study suggested that CD80/CD11b might be helpful to distinguish the CML LSCs in patients' bone marrow. It may be possible to collect a very small number of CML LSCs in CML patients under the TKI treatment using these antibodies and characterize the residual LSCs. Citation Format: Huixin Li, Shunichiro Yasuda, Satoru Aoyama, Chenyang Zhang, Yohei Kawano, Miyuki Azuma, Norihiko Kawamata. CD80 can be the marker of chronic myeloid leukemia stem cells and regulated by BCR-ABL signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3117.

CXCL12-Expressing Bone Marrow Niches Differentially Regulate CML Compared to Normal Stem Cells

CXCL12 plays a major role in hematopoietic stem cells (HSC) localization to regulatory niches in the bone marrow microenvironment (BMM). Previous studies indicate that CXCL12 deletion from Prx+ mesenchymal stem cells (MSC) reduces murine HSC numbers, and from Tek+ endothelial cells (EC) modestly reduces their repopulating activity. In contrast, CXCL12 deletion from Ocn+ osteoblasts or Osx+ osteoprogenitors does not affect HSC function (Greenbaum et al. Nature. 2013 Mar 14;495(7440):227-30; Ding et al. Nature. 2013 Mar 14;495(7440):231-5.). These studies identified MSC and endothelial cells as important HSC niche components. However, niche cells for CML leukemic stem cells (LSC) remain poorly characterized. To evaluate the contribution of CXCL12-expressing populations to LSC regulation, we transplanted normal and CML BM cells (CD45.1/2+; 2*106/mouse) into CXCL12f/f (loxP sites flanking exon 2) mice crossed with Tek-Cre and Prx-Cre mice ( reported in ASH 2016, Blood, Abstract: 26) . We showed that CXCL12 deletion from Prx+ MSC resulted in reduction in normal HSC (LSK Flt3- CD150+ CD48- cells) numbers and function compared to controls, but did not observe significant reduction of HSC with CXCL12 deletion from Tek+ EC. Interestingly, transplantation of CML cells into mice with CXCL12 deleted from Prx+ MSC led to reduced survival compared to control mice associated with increased WBC counts, BM cellularity, LSC numbers, and secondary repopulation capacity. In contrast, CXCL12 deletion from Tek+ cells led to enhanced survival, associated with reduced BM cellularity and reduced LSC numbers. In further studies, we transplanted mixtures of equal numbers of normal and CML cells into Prx-Cre and Tek-Cre mice, and found a marked growth advantage of LSC compared with HSC in Prx-Cre mice, and a growth advantage of HSC over LSC in Tek-Cre knockout mice. We also show that both HSC and LSC demonstrate increased cell cycling via EdU labeling. Consistent with this, we found that Nilotinib treatment leads to significantly greater reduction in BM cellularity, LSC numbers and secondary repopulation potential in Prx-Cre mice, compared to Tek-Cre or WT mice. To evaluate the effects of CML on CXCL12-expressing BMM cells, we crossed CXCL12GFP (GFP reporter knocked into the CXCL12 locus) and SCL-tTA-BCR-ABL mice to generate CXCL12GFP-BCR-ABL mice. CML development lead to significantly reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) but increased GFP+ endothelial cells (CD45-Ter119-CD31+), and within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased compared to WT mice. To further understand niche mediated interactions that regulate LSC fate, we studied gene expression profiles of LSC from mice following Cre-mediated targeting of CXCL12, compared to LSC from Cre-negative mice. Our preliminary analysis indicates significant enrichment of genes associated with cell cycle progression and Myc activation, and negative enrichment of genes related to TGF-b, STAT3, and Polycomb Repressive Complex 2 (PRC2) activity in LSC from Prx1+Cre CXCL12f/f mice. Reduced TGF-b, STAT3 and PRC2/EZH2 activity represent potential mechanisms for regulation of CML LSC maintenance and/or TKI resistance, and may potentially contribute to Prx1+ MSC-mediated LSC regulation. We conclude that CXCL12+ BM MSC inhibit CML development, maintain LSC quiescence, and enhance TKI resistance, while CXCL12+ EC enhance LSC maintenance and disease development. Leukemia associated reduction in CXCL12+MSC and increase in CXCL12+EC may enhance CML development. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM MSC and EC for CML LSC compared with normal HSC. DisclosuresNo relevant conflicts of interest to declare.

Identification of key candidate targets and pathways for the targeted treatment of leukemia stem cells of chronic myelogenous leukemia using bioinformatics analysis.

BackgroundChronic myelogenous leukemia (CML) is a myeloproliferative neoplasm that arises from the acquisition of constitutively active BCR‐ABL tyrosine kinase in hematopoietic stem cells. The persistence of bone marrow leukemia stem cells (LSCs) is the main cause of TKI resistance and CML relapse. Therefore, finding a key target or pathway to selectively target LSCs is of great significance for the thorough treatment of CML.MethodsIn this study, we aimed to identify key microRNAs, microRNA targets and pathways for the treatment of CML LSCs by integrating analyses of three microarray data profiles. We identified 51 differentially expressed microRNAs through integrated analysis of GSE90773 and performed functional gene predictions for microRNAs. Then, GSE11889 and GSE11675 were integrated to obtain differentially expressed genes (DEGs), and the overlapping DEGs were used as models to identify predictive functional genes. Finally, we identified 116 predictive functional genes. Clustering and significant enrichment analysis of 116 genes was based on function and signaling pathways. Subsequently, a protein interaction network was constructed, and module analysis and topology analysis were performed on the network.ResultsA total of 11 key candidate targets and 33 corresponding microRNAs were identified. The key pathways were mainly concentrated on the PI3K/AKT, Ras, JAK/STAT, FoxO and Notch signaling pathways. We also found that LSCs negatively regulated endogenous and exogenous apoptotic pathways to escape from apoptosis.ConclusionWe identified key candidate targets and pathways for CML LSCs through bioinformatics methods, which improves our understanding of the molecular mechanisms of CML LSCs. These candidate genes and pathways may be therapeutic targets for CML LSCs.

Quinacrine Depletes BCR-ABL and Suppresses Ph-Positive Leukemia Cells

Drug resistance to TKIs and the existance of CML leukemia stem cells is an urgent problem. In this study, we demonstrate that quinacrine (QC) induces apoptosis in BCR-ABL positive CML and acute lymphoblastic leukemia (ALL) cells. Interestingly, QC inhibits the colony formation of primary CD34+ progenitor/stem leukemia cells from CML patients. QC targets RNA polymerase I, which produces ribosomal (r)RNA, involving in protein translation process. Also, QC treatment prolongs CML-like mice survival and inhibits K562 tumor growth in vivo. In conclusion, we demonstrate that QC depletes BCR-ABL protein and suppresses Ph-positive leukemia cells in vitro and in vivo.

SIRT1 Mediates Enhanced Mitochondrial Oxidative Phosphorylation in Chronic Myelogenous Leukemia Stem Cells

Despite the success of BCR-ABL tyrosine kinase inhibitors (TKIs) in inducing remission and prolonging survival of CML patients, cures remain elusive because of persistence of primitive leukemia stem cells (LSC). We have reported that SIRT1, a NAD+ dependent deacetylase, is overexpressed in CML LSC and contributes to their maintenance and TKI resistance, related at least in part to increased p53 acetylation and transcriptional activity (Li et.al; Cancer Cell 2012). However, these studies using RNAi and pharmacological inhibitors were limited by possible off-target effects and limited duration of exposure. Therefore, to definitively delineate the role of SIRT1 in CML development, we developed a conditional SIRT1 knockout mouse model by crossing SIRT1 floxed mice (SIRT1fl/fl) with the Mx1-Cre strain to delete SIRT1 in hematopoietic stem cells (HSC), and crossed with transgenic BCR-ABL mice (BA Mx1-Cre SIRT1fl/fl). SIRT1 deletion in normal HSC did not inhibit normal hematopoiesis. In contrast, SIRT1 deletion in LSC profoundly inhibited the development of CML, and SIRT1-deleted CML mice did not develop evidence of morbidity and demonstrated significantly lower leukocytosis, neutrophilia and splenomegaly, and markedly improved survival, compared to controls (Fig-1). CML development in the SCL-tTA/BCR-ABL mouse model is associated with reduction in BM stem cells, and increase in stem/progenitor cell populations in the spleen. Splenic stem and progenitor populations were significantly decreased in SIRT1-deleted mice, whereas BM LTHSC were increased, indicating reversal of alterations associated with CML. SIRT1 deletion was associated with a significant decrease in LTHSC in G0 phase and increase in LTHSC in G1 phase of cell cycle. SIRT1-deleted LTHSCs generated significantly lower WBC counts, donor cell and donor LTHSC engraftment following transplantation into wild-type recipients compared to controls, indicating reduced in vivo repopulating ability. Treatment with the BCR-ABL TKI Nilotinib (50mg/kg/day) or vehicle for 4 weeks resulted in additional suppression of CML hematopoiesis in SIRT1-deleted mice. Gene expression analysis identified significant inhibition of mitochondria and oxidative phosphorylation related genes in SIRT1-deleted CML stem cells compared to controls. We performed extracellular flux analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF analyzer to evaluate mitochondrial respiration and glycolysis in c-Kit+ stem/progenitor cells from SIRT1-deleted and control CML and normal mice. CML LSC demonstrated significantly increased ATP-linked respiration, maximal mitochondrial respiration and mitochondrial reserve capacity compared to normal HSC. SIRT1-deletion resulted in significant reduction in basal and maximal mitochondrial respiration, and in mitochondrial reserve capacity, in CML LSC (Fig-2). In contrast, SIRT1-deletion did not result in significant change in mitochondrial respiration in normal HSC, suggesting that effects of SIRT1 deletion on mitochondrial respiration were specific to CML cells. The SIRT1 substrate PGC-1α is a master regulator of mitochondrial biogenesis and activity. Treatment of CML mice with the PGC-1α inhibitor SR-18292 significantly reduced basal, ATP-linked and maximal mitochondrial respiration. Consistent with results obtained with murine LSC, SIRT1 inhibition with TV39OH resulted in significant reduction in mitochondrial respiration in CML CD34+ cells compared to vehicle controls. Treatment of CML mice with Nilotinib did not significantly alter mitochondrial respiration in LSC. Similarly TKI treatment did not affect mitochondrial respiration in CML CD34+ cells compared to vehicle controls. In conclusion, our studies definitively demonstrate that SIRT1 is required for leukemia development, and reveal its critical role in increased mitochondrial respiration in CML LSC. Increased mitochondrial metabolism is kinase-independent, and is mediated by PGC-1a. SIRT1 inhibition results in enhanced inhibition of TKI-treated LSC. Our studies demonstrate an essential role for SIRT1 in altered mitochondrial metabolism in CML LSC, and support further exploration of SIRT1 pathway inhibition for LSC targeting. DisclosuresNo relevant conflicts of interest to declare.

Deregulation of calcium homeostasis in Bcr-Abl-dependent chronic myeloid leukemia.

BackgroundChronic myeloid leukemia (CML) results from hematopoietic stem cell transformation by the bcr-abl chimeric oncogene, encoding a 210 kDa protein with constitutive tyrosine kinase activity. In spite of the efficiency of tyrosine kinase inhibitors (TKI; Imatinib), other strategies are explored to eliminate CML leukemia stem cells, such as calcium pathways.ResultsIn this work, we showed that Store-Operated Calcium Entry (SOCE) and thrombin induced calcium influx were decreased in Bcr-Abl expressing 32d cells (32d-p210). The 32d-p210 cells showed modified Orai1/STIM1 ratio and reduced TRPC1 expression that could explain SOCE reduction. Decrease in SOCE and thrombin induced calcium entry was associated to reduced Nuclear Factor of Activated T cells (NFAT) nucleus translocation in 32d-p210 cells. We demonstrated that SOCE blockers enhanced cell mobility of 32d-p210 cells and reduced the proliferation rate in both 32d cell lines. TKI treatment slightly reduced the thrombin-induced response, but imatinib restored SOCE to the wild type level. Bcr-Abl is also known to deregulate Protein Kinase C (PKC), which was described to modulate calcium entries. We showed that PKC enhances SOCE and thrombin induced calcium entries in control cells while this effect is lost in Bcr-Abl-expressing cells.ConclusionThe tyrosine kinase activity seems to regulate calcium entries probably not directly but through a global cellular reorganization involving a PKC pathway. Altogether, calcium entries are deregulated in Bcr-Abl-expressing cells and could represent an interesting therapeutic target in combination with TKI.

Tigecycline May Selectively Target Leukemic Stem Cells in CML

Abstract Tigecycline inhibits mitochondrial oxidative phosphorylation to target leukemic stem cells (LSC).

Inhibition of CML Development Following Conditional SIRT1 Deletion in Transgenic BCR-ABL Mice

▪Despite the success of tyrosine kinase inhibitors (TKIs) in treatment of CML, cures remain elusive, as primitive leukemia stem cells (LSC) are retained in patients achieving remission. Previous studies from our group have suggested that Sirtuin 1 (SIRT1) inhibition may represent a novel approach for elimination of LSCs in chronic phase CML. SIRT1 was shown to be overexpressed in CML LSCs, and SIRT1 inhibition using shRNA or a small molecule SIRT1 inhibitor selectively eliminated CML LSCs by increasing p53 acetylation and activity (Li et.al; Cancer Cell 2012). These studies were limited by possible off-target effects and limited duration of in vivo exposure. Here we used a genetic mouse model to definitively delineate the role of SIRT1 in CML development. A model for conditional SIRT1 deletion in hematopoietic stem cells was established by crossing homozygous SIRT1 exon-4 floxed (SIRT1fl/fl) mice with Mx1-Cre mice. To study the requirement of SIRT1 for development of CML, Mx1-cre SIRT1fl/fl mice were crossed with SCL-tTA/BCR-ABL mice, representing a tet-regulated inducible transgenic mouse model of CML, to generate SCL-tTA/BCR-ABL Mx1-Cre SIRT1fl/fl mice (BA Mx1-Cre SIRT1fl/fl). BA SIRT1fl/fl mice lacking Mx1-Cre were used as controls. The mice were maintained on doxycycline until CML induction. Cre mediated deletion of SIRT1 was induced by intraperitoneal pIpC injections (250µg/mouse) administered every other day for a total of 7 doses. SIRT1 knockdown was confirmed by PCR for excised exon-4 and by RT-Q-PCR. Bone marrow (BM) cells from either BA Mx1-Cre SIRT1fl/fl or controls (both CD45.2) were transplanted into irradiated (800 cGy) CD45.1 congenic recipients (2X106 cells/mouse). Cre-mediated deletion of SIRT1 was induced by pIpC injection starting at 4 weeks post-transplant, followed by withdrawal of tetracycline to induce BCR-ABL expression. Serial PB counts and phenotypic evaluation of cell types by flow cytometry (Fig 1 A-B) showed SIRT1 knockdown to have a profound effect on CML development. By 8 weeks after BCR-ABL induction, BA SIRT1fl/fl mice (n=10), showed significantly lower neutrophils (p=0.0003) and Gr-1/Mac-1 positive myeloid cells (p=0.0002) compared to control mice. Subsequently, control mice developed progressive neutrophilic leukocytosis and increasing morbidity from leukemia, whereas BA SIRT1fl/fl mice demonstrated significantly lower WBC counts, without evidence of progressive increase or morbidity (Fig 1 A). This cohort of mice continues to be followed for survival. Another cohort of BA Mx1-Cre SIRT1fl/fl mice was sacrificed at 8 weeks post pIpC injection and BCR-ABL induction to evaluate the effect of SIRT1 knockdown on stem and progenitor populations (n=6 each). SIRT1 deleted mice demonstrated significant reduction in spleen size, weight, cellularity, and myeloid infiltration (Fig 2 A-B), and in myeloid cell expansion in the BM compared to controls (p=0.002). Primitive lineage negative, Sca1 positive, c-Kit negative (LSK) cells and granulocyte-macrophage progenitors (GMP) were significantly reduced in BM and spleen of BA SIRT1 deletedmice compared to control mice, whereas megakaryocyte-erythrocyte progenitors (MEP) were increased (Fig 3 A-B). Long term hematopoietic stem cells (LTHSC) in the BM are reduced following CML development. The percentage and number of LTHSC were significantly increased in SIRT1 deletedmice compared to control mice (Fig 3C-D). We also evaluated the effect of SIRT1 deletion on normal hematopoiesis by studying Mx1-Cre SIRT1fl/fl mice lacking BCR-ABL. SIRT1fl/fl mice without Mx1-Cre were studied as controls. Mx1-Cre SIRT1fl/fl and control mice were treated with pIpC to induce SIRT1 deletion. SIRT1deletedmice did not show significant alteration in blood counts, but demonstrated significantly higher LSK and LTHSC numbers in BM compared to control mice. Upon secondary transfer, recipients of BM from SIRT1deleted mice showed a modest increase in donor cell engraftment at 12 weeks compared to controls (90.8% (83.2-92.2%) vs 83.6% (75.8-86.7%); p=0.001). We conclude that genetic deletion of SIRT1 markedly inhibits all aspects of CML development in transgenic BCR-ABL mice, without impairing normal hematopoiesis. These observations demonstrate a critical role for SIRT1 in leukemia development, and support further evaluation of SIRT1 as a therapeutic target in CML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of CML Stem Cells with an Alkaloid That Reduces β-Catenin

Although the majority of CML patients initially respond positively to BCR-ABL tyrosine kinase inhibitors (TKIs), they fail to eradicate the leukemia stem cells (LSCs) from which the disease arises. Only a minority of patients is able to discontinue TKI therapy, presumably due to the survival of LSCs. Therefore, the development of new therapeutics which ablate CML stem cells through a non-TKI, BCR-ABL independent pathway is needed.The Wnt/β-catenin pathway has been identified as an LSC survival pathway which provides proliferative signals and controls the stability of BCR-ABL1 through the increased expression of β-catenin. While previous research has demonstrated that Wnt/β-catenin is necessary for the survival and self-renewal of all CML cells and LSCs, it is not essential for maintenance of normal hematopoietic stem cells (HSCs).Tetrandrine (ES-3000, TET) is a natural product alkaloid used clinically in China as an analgesic and an anti-inflammatory. Its known mechanism of action is the inhibition of voltage-gated calcium channels and calcium activated big potassium (BK) channels which are commonly overexpressed in malignancies. However, TET has recently been demonstrated to inhibit the Wnt/β-catenin pathway resulting in a reduced expression of β-catenin, putatively through the inhibition of CaMKII-γ activation. This study investigated the efficacy of TET in models of CML stem cells.To demonstrate that TET can reduce β-catenin in leukemic cells, an in vitro assay with leukemic K562 cells was performed. Cells were exposed to TET for 24 hours at concentrations between 0-40 μM. Cell lysates were assayed by Western blot for β-catenin and actin. The results demonstrated that TET reduces β-catenin expression in a dose dependent manner.The effectiveness of TET was tested on CML stem cells using an in vivo mouse CML model. After priming donor C57BL/6 (B6) mice with intravenous injections of 5-fluorouracil for four days, bone marrow cells were harvested from femurs and tibia, then transfected twice with retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were lethally irradiated by two doses of 550 cGy before bone marrow transplantation by intravenous injection with 5x105 cells/mouse. Blood from recipient mice was tested for disease induction one week after transduction by FACS analysis for GFP. All mice tested positive.Treatments started on day 8 after bone marrow transplantation. Mice were randomized into four groups and treated orally with vehicle [3x/day, 2x], imatinib [100 mg/kg, 2x/day], TET [150 mg/kg, 1x/day] or imatinib + TET [3x/day: 2x with imatinib, 1x with TET]. The results of this study demonstrated that TET given orally once a day is superior to imatinib given twice a day in inhibiting the development of both circulating leukemic cells and leukemic stem cells while the combination of TET with imatinib further improves efficacy (See Figure).To determine whether TET has efficacy on human CML stem-like cells, a colony forming cell (CFC) assay with bone marrow cells from a de-novo CML patient was performed. The bone marrow cells were treated with 10 µM (IC50) TET for 14 days. After treatment, primary and secondary colonies were grown and analyzed by qPCR to determine BCR-ABL or ABL only cells. Replating efficiency of TET treated cells was 54% compared to 67% of solvent controls. In primary colonies, 95% of colonies from solvent control cultures were BCR-ABL positive compared to 70% of colonies treated with ES-3000. In secondary colonies (representing stem-ness), the TET treatment group was negative for BCR-ABL colonies while 79% of solvent control colonies still tested positive for BCR-ABL, indicating efficacy of TET in CML stem-like cells (See Table).We conclude that TET reduces leukemic stem cells in both a murine model of CML and a CFC assay which demonstrates its potential for development as an adjuvant therapy for CML patients demonstrating a lack of optimal response to TKIs, alone. [Display omitted] [Display omitted] DisclosuresMichaels:Escend Pharmaceuticals, Inc.: Equity Ownership. Bates:Escend Pharmaceuticals, Inc.: Equity Ownership.

Notch Pathway Activation Targets Leukemic Stem Cells in Chronic-Phase Chronic Myeloid Leukemia (CP-CML)

The phenomenon of disease persistence in CP-CML under therapy with ABL kinase inhibitors suggests BCR-ABL-independent mechanisms are being exploited to sustain the survival of leukemic stem cells (LSC). Many self-renewal pathways have been implicated in their survival. The role of Notch signalling has yet to be fully elucidated, with conflicting reports suggesting a variety of roles in myeloid disease. The aims of this project were (1) to assess the expression and functional role of the Notch pathway in primary CD34+CP-CML cells, and (2) to assess differences in the functional role between stem and progenitor subpopulations in disease maintenance and progression. We hypothesized that Notch activation would have different functional effects dependent on disease phase and cell maturity.Extensive analysis of existing microarray datasets, GSE47927 and E-MTAB2481, comparing CP-CML LSCs to normal HSCs, showed that the Notch pathway was downregulated in CP-CML (p=0.05, both). To validate these results, using Fluidigm technology, mRNA expression of untreated CD34+ CP-CML cells (n=24) was compared to normal bone marrow CD34+ cells (n=5). The Notch pathway was silenced in untreated CP-CML, as evidenced by downregulation of Notch target genes, HES1 (p<0.0001) and HIF1A (p=0.0029). The Notch receptor, NOTCH2, was significantly upregulated (p<0.0001) in the same samples. This was confirmed with immunofluorescence (IF) of the full-length receptor (n=5). The activated form of the Notch receptor, val1744, was absent on CD34+CP-CML cells by either IF or Western blotting (n=3), nor was there any significant change in functional analyses of CP-CML cells treated with the pharmacological Notch inhibitor, DAPT (n=5). These results support our hypothesis that the pathway is silenced in CP-CML.As CD34+ CP-CML cells abundantly express NOTCH2, we investigated whether the pathway could be reactivated through exogenous stimulation upon ligand binding. An OP9 co-culture system was utilized to allow for overexpression of Notch ligands, DLL1 or JAG1, using OP9GFP as control stroma. Compared to non-stromal conditions, culturing over 7 days on OP9GFP led to a significant increase in growth (p=0.02, n=5). The overexpression of JAG1 led to a further increase compared to OP9GFP (p=0.002). We confirmed that activation of the pathway was through JAG1, and not DLL1, by utilizing DAPT, where we observed a significant decrease in live cell counts within the OP9GFP (p=0.01) and OP9JAG1 (p=0.018) experimental arms. Upregulation of the pathway was confirmed with increased expression of HES1 at the gene level (n=4, p=0.001), andval1744 protein by IF (n=5).CP-CML samples (n=3) were FACS-sorted into Lin-CD34+38+ and Lin-CD34+38- populations to assess for functional variation in stem and progenitor populations. Following 7 days in culture, live cell counts showed a significant increase in Lin-CD34+38+ cells on OP9JAG1 co-culture (p=0.04). There was a decrease in cell growth of Lin-CD34+38- cells in the same experimental conditions (p=0.02), with an associated increased trend in late apoptotic cells (ns). We hypothesized that Notch activation would have a toxic influence on an immature CML LSC population. LTCIC assays showed a significant decrease in colonies for CD34+38-cells co-cultured with OP9JAG1 for 7 days (p<0.05, n=8).In myeloid BP (n=4), in similar experiments, sorted Lin-CD34+38+ and Lin-CD34+38- populations did not demonstrate this functional profile of Notch activation, despite an upregulation in NOTCH2 receptor expression compared to normal (n=11) (p=0.0035). To investigate this further, we sorted CP (n=12), myeloid BP (n=11), and lymphoid BP (n=5) into stem and progenitor populations, before undertaking quantitative PCR of 90 self-renewal gene components of Wnt, Notch, Hedgehog, and BMP pathways. The self-renewal pathways were highly deregulated between CP and BP. The differential gene expression in lymphoid BP was comparable to myeloid BP. There was statistically significant upregulation in Wnt components in myeloid BP compared to CP, particularly TCF7(p=0.0011). We hypothesize that Wnt upregulation is preventing Notch activation in myeloid BP-CML.Taken together, our results identify that Notch activation may be a therapeutic approach to target CP-CML LSCs. However, this cannot be translated to myeloid BP, highlighting the complexity of self-renewal pathway interaction. DisclosuresWheadon:GlaxoSmithKline: Research Funding. Holyoake:Novartis: Honoraria, Research Funding; Bristol Myers Squib: Honoraria, Research Funding. Copland:ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Honoraria; Amgen: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Identification and functional characterization of the miRNA-gene regulatory network in chronic myeloid leukemia lineage negative cells.

Chronic myeloid leukemia (CML) is maintained by leukemic stem cells (LSCs) which are resistant to the existing TKI therapy. Hence a better understanding of the CML LSCs is necessary to eradicate these cells and achieve complete cure. Using the miRNA-gene interaction networks from the CML lin(−) cells we identified a set of up/down-regulated miRNAs and corresponding target genes. Association studies (Pearson correlation) from the miRNA and gene expression data showed that miR-1469 and miR-1972 have significantly higher number of target genes, 75 and 50 respectively. We observed that miR-1972 induces G2-M cell cycle arrest and miR-1469 moderately arrested G1 cell cycle when overexpressed in KCL22 cells. We have earlier shown that a combination of imatinib and JAK inhibitor I can significantly bring down the proliferation of CML lineage negative cells. Here we observed that imatinib and JAK inhibitor I combination restored the expression pattern of the down-regulated miRNAs in primary CML lin(−) cells. Thus effective manipulation of the deregulated miRNAs can restore the miRNA-mRNA networks that can efficiently inhibit CML stem and progenitor cells and alleviate the disease.

Abstract 3338: Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche

Abstract Introduction Pacritinib, a potent clinical small molecule inhibitor of JAK2, it also suppresses signaling through wild-type and mutant FLT3, IRAK1, and CSF1R. Pacritinib does not cause marrow suppression and has demonstrated single agent activity preclinically in myelofibrosis and other myeloid neoplasms including AML and CMML. Stromal protection was not observed. However, the capacity of pacritinib to eradicate therapy resistant leukemia stem cells (LSC), residing in the bone marrow niche, had not been examined. Thus, we investigated the impact of pacritinib alone or in combination with standard of care therapy on primary blast crisis chronic myeloid leukemia (BC CML), myelofibrosis (MF) and AML LSC survival and self-renewal in a stem cell supportive niche. Methods Genetically engineered mouse bone marrow fibroblasts producing human SCF, IL3 and G-CSF were used as stromal monolayers to support LSC survival and self-renewal. Human primary CD34+ cells were selected from BC CML (n = 5), MF (n = 5) and relapsed AML (n = 4) before and after clinical treatment with azacitidine. As a control, CD34+ cells from age matched normal bone marrow (a-NBM, n = 4) were used for the co-culture. Survival and self-renewal of the cells were investigated by colony forming and replating assays. Pacritinib was used at concentrations ranging from 10 to 50 nM alone and in combination with 1 nM dasatinib. Results Pacritinib alone induced dose-dependent inhibition of self-renewal in a-NBM, AML, MF and CML-BC, with the optimal concentration of 20nM leading to IC50 diversity in the response between normal and leukemia progenitors. AML and MF responded uniformly and inhibition reached 50% at 10nM concentration. BC CML cells were more divergent: 40% demonstrated &amp;gt;50% inhibition, in another 40% it was 20-50% and in 20% inhibition was &amp;lt;20%. Combined treatment with the low dose of dasatinib and pacritinib doses of 10 or 20 nM resulted in a significant (p&amp;lt;0.001, Anova) difference in self-renewal of all BC CML cells, raising a possibility of an additive/synergistic mechanisms. AML cells collected before and after clinical treatment with azacitidine uniformly showed a significant decrease in self-renewal starting with 10 nM pacritinib alone and combined treatment with dasatinib did not enhance the inhibition, suggesting a prospect of using pacritinib as a single agent in the treatment of relapsed AML. Conclusions Together these data indicate that possibly through inhibition of CSF1 and IRAK1 signaling in addition to suppression of JAK2, even in the presence of a LSC supportive niche, readily clinically achievable low nM concentrations of pacritinib alone are effective in reducing self-renewal of MF and relapsed AML. However, a combination of dasatinib and pacritinib is required to eliminate self-renewing LSC in BC CML with minimal toxicity toward normal progenitors. Targeting niche-dependent signaling could represent a robust avenue for treatment of refractory myeloid leukemia. Citation Format: Larisa Balaian, Anna Kulijian, Edward D. Ball, Catriona H.M Jamieson. Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3338.

Immunological Analyses of Leukemia Stem Cells.

Traditionally, the intracellular localization and expression levels of specific proteins in CML Leukemia stem cells (LSCs) have been evaluated by fluorescence immunohistochemistry (FIHC). More recently, Duolink(®) in situ PLA technology has opened up a new and more quantitative way to evaluate signal transduction, posttranslational modification, and protein-protein interaction at the single-stem-cell level. This novel methodology, which employs two antibody-based probes, has already increased our understanding of the biology of the rare CML LSC population. In the future, the use of this approach may contribute to the development of novel therapeutics aimed at eradicating CML LSCs in CML patients.

Identification of a New AHI-1-Mediated-PP2A-Beta-Catenin-BCR-ABL-JAK2 Complex Modulating Resistance of CML Stem/Progenitor Cells to Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitor (TKI) therapies have had a major impact on treatment of early phase CML patients. However, TKI monotherapies are not curative and initial and acquired resistance remain challenges. Particularly, leukemic stem cells (LSCs) are less responsive to TKIs and are a critical target population for TKI resistance. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is highly upregulated in LSCs and interacts with multiple kinases, including BCR-ABL and JAK2. We further showed that AHI-1-mediated complexes contribute to enhanced transforming activity of BCR-ABL and drug resistance of LSCs, suggesting AHI-1 as a new therapeutic target in CML. By screening the Prestwick Chemical Library, we have recently identified a specific growth inhibitory compound that potentially targets AHI-1: Cantharidin (CAN), an inhibitor of protein phosphatase 2A (PP2A). PP2A is a family of serine/threonine phosphatases that regulate cell signaling cascades involved in proliferation and cell cycle control of cancer cells. Evidence suggests the dysfunction of specific PP2A protein complexes is primarily responsible for the changes that lead to cell transformation. It was also reported that PP2A activity is downregulated in CML cells, particularly in blast crisis CML, due to overexpression of SET, and that the use of a PP2A activator inhibits the growth of CML cells. Interestingly, we have now demonstrated that CAN inhibits the growth of AHI-1-transduced BCR-ABL+ cells by about 30% compared to control cells, but this effect was significantly enhanced to 93% with the addition of imatinib (IM). As well, AHI-1-suppressed cells were more sensitive to CAN treatment. PP2A is highly expressed in K562 and IM-resistant K562 cells and combination treatment with CAN and TKI prevents growth and induces apoptosis in these cells more effectively than single treatments (2-3 fold, p<0.001). The combination also greatly reduced colony formation (CFC) of CD34+ CML cells, but CAN also inhibited CFCs of CD34+ normal bone marrow (BM) cells. To overcome toxicity issues, we have recently evaluated new, pre-clinically validated PP2A inhibitors, LB100 and LB102. These specifically suppress PP2A activity up to 75% in CML cells, as determined by immunoprecipitation (IP) phosphatase assays, and inhibit growth of these cells. Importantly, PP2A inhibitors plus TKIs significantly reduced the yield of colonies from CD34+ CML stem/progenitor cells compared to any single agent or combination of TKIs and this enhanced effect was most pronounced on cells from IM nonresponders (p<0.05). New PP2A inhibitors are also much less toxic to CD34+ normal BM cells compared to CAN (2-3 fold, p<0.001). Long-term culture-initiating cell assays showed that more primitive cells were eliminated to a greater extent by combination treatments. Moreover, the combination resulted in strong synergy in IM-insensitive cells (CI value <0.5). Notably, cell cycle analysis showed that treatment with LB100/LB102 alone induces a shift from G1 to G2/M phase (3-fold, p<0.05). A similar shift in the cell population was observed after combination treatment with IM, suggesting that the G2/M phase arrest is solely due to PP2A inhibition. Confocal microscopy showed the G2/M arrest, leading to mitotic catastrophe. Mechanistically, we have identified the PP2A subunit B (PR55a) as an AHI-1 interacting protein using IP/mass spectrometry; this was further confirmed in AHI-1-transduced cells by IP-Western analysis. Most interestingly, Western blot analysis showed that the combination treatment not only inhibits tyrosine phosphorylation of BCR-ABL, JAK2, STAT5, AKT and P38, but also significantly inhibits protein expression of these proteins compared to single agents. Inhibition of protein expression of AHI-1 and β-catenin by the combination was also observed and a new protein interaction between AHI-1 and β-catenin was identified. Moreover, the combination directly affects PP2A-mediated β-catenin dephosphorylation and BCR-ABL-mediated phosphorylation of β-catenin in CML cells, which leads to protein degradation of multiple proteins. In summary, we have uncovered new AHI-1-PP2A (PR55a) and AHI-1- β-catenin interactions and simultaneously targeting both BCR-ABL and PP2A activities in CML LSCs may offer an important new therapeutic possibility, through destabilization of the protein-protein interactions mediated by AHI-1. DisclosuresNo relevant conflicts of interest to declare.

A New AHI-1-DNM2-BCR-ABL Complex Regulates Leukemic Properties of Primitive CML Cells through Mediation of Cellular Endocytosis and ROS-Induced Autophagy

Tyrosine kinase inhibitor (TKI) therapies have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells (LSCs) remain problematic. Improved treatment approaches to specifically target key molecular elements active in CML LSCs are needed. One candidate is the adaptor protein AHI-1 (Abelson helper integration site-1), an oncogene that is highly deregulated in LSCs. An AHI-1-mediated protein complex containing BCR-ABL and JAK2 has been shown to modulate transforming activity and TKI-response/resistance of CML LSCs. We have recently identified the large GTPase Dynamin-2 (DNM2) as another AHI-1 interacting protein using the AHI-1 SH3 domain as protein "bait" in immunoprecipitation (IP)/mass spectrometry. DNM2 plays key roles in the regulation of trafficking processes such as endocytosis, and is activated through tyrosine phosphorylation. However, its role in CML pathogenesis is unknown. We have now demonstrated that transcript levels of DNM2 are significantly increased in treatment-naive CD34+ cells from CML patients who were classified retrospectively, after Imatinib (IM) therapy, as IM-responders (n=11) and IM-nonresponders (n=15) in comparison to CD34+ normal bone marrow cells (n=7, p=0.013 and 0.037). In particular, DNM2 is more highly expressed in CML stem-enriched cells (lin-CD34+CD38-) and progenitor cells (lin-CD34+CD38+) than more mature cells (lin+CD34-, 2-fold). Interestingly, BCR-ABL+ human cells with stable knockdown of DNM2 exhibited significantly reduced cell growth and increased apoptosis as compared to control cells. They also showed enhanced sensitivity to single TKIs or MitMAB (DNM2 inhibitor) and these effects were greatly enhanced by combination treatments (2-3 fold). Mechanistically, co-IP and confocal co-localization analysis with mutant forms of AHI-1 and DNM2 (HA-AHI-1, HA-AHI-1 SH3Δ, Myc-DNM2 and Myc-DNM2 PRDΔ) in 293T cells indicated that the PRD domain of DNM2 is mainly responsible for the interaction between the SH3 domain of AHI-1 and DMN2. More importantly, we identified a new protein interaction between DNM2 and BCR-ABL in both BCR-ABL and BCR-ABL/AHI-1 co-transduced hematopoietic cells using co-IP/Western analysis; this interaction is enhanced in BCR-ABL/AHI-1 co-transduced cells. Moreover, DNM2 phosphorylation was decreased upon IM treatment in BCR-ABL-transduced cells, but remained unchanged in control and T315I mutant cells, suggesting that DNM2 is a direct target of BCR-ABL. Interestingly, we further observed that AHI-1 co-localizes with EEA-1 (early endosome marker) and LAMP-1 (late endosome marker) in cells co-transfected with full-length AHI-1 and DNM2, but not in AHI-1 and DNM2 mutant cells. Using transferrin uptake assays, increased transferrin signals were observed in BCR-ABL/AHI-1 co-transduced cells compared to BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while transferrin signals were much lower in DNM2-knockdown cells than control cells. These results suggest that the AHI-1-DNM2-BCR-ABL complex indeed improves the kinetics and increases efficiency of endocytosis, which may contribute to response/resistance of primitive CML cells to TKIs. This is further supported by observation of increased surface expression levels of CXCR4 in DNM2 knockdown cells, a key endocytotic target and known mediator of TKI response in CML. Moreover, reactive oxygen species (ROS) signals were found to be higher in BCR-ABL/AHI-1 co-transduced cells than BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while ROS accumulation was significantly decreased in DNM2-knockdown cells compared to control cells. More interestingly, ROS-induced autophagy was further observed in BCR-ABL/AHI-1 co-transduced cells, but reduced in DNM-2-knockdown cells, with protein expression changes in key autophagy proteins, including ULK-1, Beclin-1, LC3 and p62. To the best of our knowledge, this is the first study to implicate a new AHI-1-DNM2-BCR-ABL complex in the deregulation of endocytosis signaling and ROS production/autophagy in CML. This may play a unique and important role in the regulation of cellular properties of primitive CML cells, including their response/resistance to TKI. DisclosuresNo relevant conflicts of interest to declare.