BCR-ABL Positive Cells Research Articles

ASP210: a potent oligonucleotide-based inhibitor effective against TKI-resistant CML cells.

Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 µM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy.NEW & NOTEWORTHY Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells.

Asciminib Maintains Antibody-Dependent Cellular Cytotoxicity against Leukemic Blasts.

B cell acute lymphoblastic leukemia (B-ALL) is characterized by an accumulation of malignant precursor cells. Treatment consists of multiagent chemotherapy followed by allogeneic stem cell transplantation in high-risk patients. In addition, patients bearing the BCR-ABL1 fusion gene receive concomitant tyrosine kinase inhibitor (TKI) therapy. On the other hand, monoclonal antibody therapy is increasingly used in both clinical trials and real-world settings. The introduction of rituximab has improved the outcomes in CD20 positive cases. Other monoclonal antibodies, such as tafasitamab (anti-CD19), obinutuzumab (anti-CD20) and epratuzumab (anti-CD22) have been tested in trials (NCT05366218, NCT04920968, NCT00098839). The efficacy of monoclonal antibodies is based, at least in part, on their ability to induce antibody-dependent cellular cytotoxicity (ADCC). Combination treatments, e.g., chemotherapy and TKI, should therefore be screened for potential interference with ADCC. Here, we report on in vitro data using BCR-ABL1 positive and negative B-ALL cell lines treated with rituximab and TKI. NK cell activation, proliferation, degranulation, cytokine release and tumor cell lysis were analyzed. In contrast to ATP site inhibitors such as dasatinib and ponatinib, the novel first-in-class selective allosteric ABL myristoyl pocket (STAMP) inhibitor asciminib did not significantly impact ADCC in our settings. Our results suggest that asciminib should be considered in clinical trials.

Topolink™: Advancing Cancer Genomics with a High-Resolution, High-Throughput Approach for Low Sequencing and Highly Sensitive Detection of Structural Variants

Despite numerous technological advances, including the widespread adoption of massively parallel genome sequencing, many clinically relevant cancer driver mutations go undetected. Additionally, even with the most comprehensive cancer profiling using a combination of whole genome and whole transcriptome sequencing, a driver mutation goes undetected in approximately 20% of cancers, making targeted treatment of these patients challenging. The reason for this gap in understanding is presumably two-fold: 1) current technologies do not have the required sensitivity for detection of the causative variants; or 2) causative variants are epigenetic or regulatory in nature, meaning the driving alteration does not result in a change to the core DNA sequence. An explanation for 1) is that structural variants (SVs) are particularly challenging to detect using shotgun-based approaches, since these depends on the presence of specific chimeric molecules within the library that directly span or bridge the breakpoint. Long-read technologies circumvent this limitation but have significant drawbacks with respect to cost and strict sample requirements. Another possibility is that standard sequencing - which results in hundreds or thousands of variants of unknown significance (VUS) - may in fact contain causative variants, but our understanding of function of these variants is limited. For 2), it is increasingly appreciated that epigenetic and chromatin topological features are fundamental in the gene regulation and disease. To address this gap in our understanding of cancer, we utilized TopoLink™ proximity ligation library protocol that yields high-quality, high-resolution, unbiased HiC libraries and that can be performed in under 8 hours. To our knowledge, TopoLink™ is the first and only assay of its kind. Proximity ligation offers enhanced sensitivity for detection of SVs, and the restriction enzyme-free digestion method ensures the uniform coverage needed for accurate detection of single nucleotide variations (SNVs) and copy number variants (CNVs). To our knowledge, TopoLink™ is the first and only assay combine the speed, throughput, and unbiased primary base coverage of WGS with the improved detection of large SVs in Hi-C data. Detection of SVs provides a critical basis for personalized therapies in hematological cancers. To test the ability of TopoLink™ to detect clinically relevant SVs, we used the BCR-ABL1 positive CML cell line K562 to determine the limit of detection of interchromosomal translocations. In addition, we calculated the sensitivity of SV detection relative to current industry-leading sequencing technologies using the breast carcinoma cell line HCC1187 gold standard. We noted enhanced sensitivity for SV detection for TopoLink™ relative to both long-read technologies and WGS. Using TopoLink™, sensitivity was greater than 95% at a genome coverage of less than 1x, whereas both long-read and WGS sensitivity drops below 95% sensitivity at approximately 10x genome coverage. Similarly, using hybridization capture of BCR-ABL1 in a TopoLink™ library of K562 cells reduced the required sequencing burden by 10-fold relative to standard shotgun approaches. Importantly, we demonstrate that TopoLink™ libraries maintain topological features consistent with biologically relevant topologically-associated domains (TADs) and chromatin loops, thus enabling insight into novel epigenetic cancer drivers. Therefore, we demonstrate TopoLink™ proximity ligation libraries as a complementary technique that offers enhanced sensitivity of clinically relevant structural variants, with the added benefit of improving discovery of novel epigenetic mechanisms. Finally, the reduced sequencing costs needed to detect clinically relevant SVs allows for improved diagnostics and personalized medicine in a clinical or translational research setting.

Synergistic cytotoxicity effect of the combination of chitosan nanoencapsulated imatinib mesylate and quercetin in BCR-ABL positive K562 cells.

Intolerable side effects and resistance to chemotherapeutic drugs have encouraged scientists to develop new methods of drug combinations with fewer complications. This study aimed to investigate the synergistic effects of quercetin and imatinib encapsulated in chitosan nanoparticles on cytotoxicity, apoptosis, and cell growth of the K562 cell line. Imatinib and quercetin were encapsulated in chitosan nanoparticles and their physical properties were determined using standard methods and SEM microscope images. BCR-ABL positive K562 cells were cultured in a cell culture medium, cytotoxicity of drugs was determined by MTT assay and the effects of nano drugs on apoptosis in cells were investigated by Annexin V-FITC staining. The expression level of genes associated with apoptosis in cells was measured by real-time PCR. The IC50 for the combination of the nano drugs at 24 and 48 hr was 9.324 and 10.86 μg/ml, respectively. The data indicated that the encapsulated form of drugs induced apoptosis more effectively than the free form (P<0.05). Moreover, the synergistic effect of nano drugs in statistical analysis was proved (P=0.001). The combination of nano drugs resulted in the caspase 3, 8, and TP53 genes upregulation (P=0.001). The results of the present study showed that the encapsulated form of imatinib and quercetin nano drugs with chitosan has more cytotoxicity than the free form of the drugs. In addition, the combination of imatinib and quercetin as a nano-drug complex has a synergistic effect on the induction of apoptosis in imatinib-resistant K562 cells.

Ferrocene modified analogues of imatinib and nilotinib as potent anti-cancer agents.

The unique features of ferrocene and the need for development of targeted anticancer drugs inspired the design, synthesis and biological evaluation of ferrocenyl modified tyrosine kinase inhibitors by replacing the pyridyl moiety in imatinib and nilotinib generalized structures with a ferrocenyl group. A series of seven new ferrocene analogues were synthesized and evaluated for their anticancer activity in a panel of bcr-abl positive human malignant cell lines using imatinib as a reference drug. The metallocenes exhibited a dose-dependent inhibition on malignant cell growth with varying antileukemic activity. The most potent analogues were compounds 9 and 15a showing comparable or even superior efficacy to the reference. Their cancer selectivity indices suggest a favorable selectivity profile, indicating a 250 times higher preferential activity of 15a towards malignantly transformed K-562 cells and an even twice greater one (500) of 9 in the LAMA-84 leukemic model as compared to the normal murine fibroblast cell line.

ASP210 - a Potent Oligonucleotide-Based Inhibitor for Combating Both BCR-ABL1 Dependent and Independent TKI-Resistant CML Cells

Background: Tyrosine kinase inhibitors (TKIs) have remarkably changed the management of Ph+ chronic myeloid leukemia (CML) and have dramatically contributed to substantial prolongation of survival rates and superior quality of life. However, long-term clinical experience with TKIs shows that despite the apparent therapeutic benefit, ~35% of CML patients exhibit primary resistance or intolerance to TKIs, and ~25% of those treated is forced to switch TKI at least once during therapy due to acquired resistance. Mutations in the kinase domain of BCR-ABL1 are the most comprehensively described mechanism of TKI-resistance but fail to explain ~40% of resistant cases. Nevertheless, due to the availability of several TKIs, the issue of TKI-resistance has de facto reduced to the mutation T315I and BCR-ABL1 independent resistance. TKI-resistance thus represents a persistent clinical problem to be solved and ultimately calls for alternative treatment solutions for CML patients with leukemic clones escaping TKI therapy. Oligonucleotide therapeutics have generated high expectation stemming from their potency and the field has seen great advancements leading to an apparent progress in clinical trials. Given the vastly different molecular target compared to protein inhibitors (RNA versus protein) as well as principally different mechanism of action, oligonucleotide therapeutics might represent rational and potent tools to combat TKI-resistant CML cells. Progress beyond the state-of-the art: The design of standard oligonucleotides is relatively straightforward and is sequence driven. However, the promiscuity of oligonucleotides places severe limitations on this approach since sequence-dependent off-target effects deteriorate their toxicological profile. Unconventionally, we considered additional parameters during the design of an oligonucleotide-based inhibitor, including steric and thermodynamic aspects of its binding to the target RNA, which significantly improved its safety. Here we report on a potent, safe, and highly specific oligonucleotide-based modality against BCR-ABL1 mRNA, ASP210, which effectively induces apoptosis in both BCR-ABL1 dependent and independent TKI-resistant CML cells. Methods:BCR-ABL1 positive cell lines MOLM-7 a CML-T1, both sensitive and resistant to TKIs imatinib and dasatinib were used to investigate the efficacy of ASP210, a biotinylated 34mer phosphorothioate DNA with an internal poly(ethylene glycol) linker. Briefly, MOLM-7 and CML-T1 cells and their resistant sub-lines were incubated with defined concentrations of ASP210 ranging between 0.25-5.0 µM for 10 days (applied once daily). Cell viability was monitored by trypan blue staining every day. The results were normalized to control, i.e., to untreated cells on the respective day. Of note, MOLM-7 TKI-resistant cells did not possess any mutation in the kinase domain, while both resistant sub-lines derived from CML-T1 harbored the clinically relevant mutation T315I. BCR-ABL1 negative cell line HL-60 was used as target negative control in the same experimental setup. Results:In vitro functional tests of ASP210 revealed its ability to induce cell death of all BCR-ABL1 positive cell types studied. A continuous decrease in the viability of ASP210-exposed cells has been observed already after 24 hours in all cell types including both TKI-sensitive CML cells and the derived TKI-resistant sub-lines irrespective of their T315I status. The efficacy of ASP210 was confirmed to be dose-dependent, with ASP210 causing a ~90% reduction of CML cells by day 5. Importantly, no effect on the viability of BCR-ABL1 negative HL-60 cells was observed (viability remained ≥95%) upon exposure to ASP210. Conclusions: The present study demonstrates the cytoreductive potential of ASP210 in terms of its ability to induce apoptosis in both BCR-ABL1 dependent and independent TKI-resistant CML cells, with no effect on BCR-ABL1 negative cells.

Thymoquinone Inhibits JAK/STAT and PI3K/Akt/ mTOR Signaling Pathways in MV4-11 and K562 Myeloid Leukemia Cells.

Constitutive activation of Janus tyrosine kinase-signal transducer and activator of transcription (JAK/STAT) and Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways plays a crucial role in the development of acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Thymoquinone (TQ), one of the main constituents of Nigella sativa, has shown anti-cancer activities in several cancers. However, the inhibitory effect mechanism of TQ on leukemia has not been fully understood. Therefore, this study aimed to investigate the effect of TQ on JAK/STAT and PI3K/Akt/mTOR pathways in MV4-11 AML cells and K562 CML cells. FLT3-ITD positive MV4-11 cells and BCR-ABL positive K562 cells were treated with TQ. Cytotoxicity assay was assessed using WSTs-8 kit. The expression of the target genes was evaluated using RT-qPCR. The phosphorylation status and the levels of proteins involved in JAK/STAT and PI3K/Akt/mTOR pathways were investigated using Jess western analysis. TQ induced a dose and time dependent inhibition of K562 cells proliferation. TQ significantly downregulated PI3K, Akt, and mTOR and upregulated PTEN expression with a significant inhibition of JAK/STAT and PI3K/Akt/mTOR signaling. In conclusion, TQ reduces the expression of PI3K, Akt, and mTOR genes and enhances the expression of PTEN gene at the mRNA and protein levels. TQ also inhibits JAK/STAT and PI3K/Akt/mTOR pathways, and consequently inhibits proliferation of myeloid leukemia cells, suggesting that TQ has potential anti-leukemic effects on both AML and CML cells.

Evaluation of anti-tumor effect of the exopolysaccharide from new cold-adapted yeast, Rhodotorula mucilaginosa sp. GUMS16 on chronic myeloid leukemia K562 cell line

Recently, the development and application of fungal exopolysaccharides (EPS) as natural biopolymers are on the rise. The present study is based on the investigation of possible antiproliferative and antioxidant activities of EPS from the Rhodotorula mucilaginosa sp. GUMS16 on BCR-ABL positive cells (K562). The cytotoxicity, colony formation assays lactate and dehydrogenase (LDH) activity were performed to assess the possible cancer cell death. To elucidate the underlying antiproliferative mechanism of the EPS, cell cycle analysis following real-time PCR (gene expression assessment) were evaluated. The results indicated that, the EPS with an IC50 dose of 1500 μg/ml, reduced the viability of K562 cells without having toxic effects on normal cells as well as decrease in size and number of colonies in EPS-treated group (p < 0.0001). The increase of LDH was 2.75 times more than the control (p < 0.0001). Gene expression revealed up- and down-regulation of apoptotic and anti-apoptotic genes in EPS group compared with the control. Moreover, the DPPH scavenging activity of the EPS in treated cells was significantly higher than the control group (p < 0.0001). Taken together, we concluded that the EPS from GUMS16 strain is able to inhibit the growth of K562 cells besides having antioxidant activities.

Bcr-Abl drives the formation of Hsp70/Bim PPI to stabilize oncogenic clients and prevent cells from undergoing apoptosis

Although tyrosine kinase inhibitors that inhibit Bcr-Abl kinase activity have shown excellent efficacy in the clinical application of CML patients, it still a challenge to discover alternative targets and novel therapies because of the emergence of Bcr-Abl-independent resistance. Most recently, Hsp70/Bim complex was revealed that driven by Bcr-Abl and testified as a specific target for CML because it folds and stabilizes many Hsp70 oncogenic substrates that mediate CML specific signaling pathways. However, the relationship between Bcr-Abl and Hsp70/Bim complex and how the chaperone complex contributes to Bcr-Abl-driven leukemogenic cells remain unclear. Herein, with the help of S1g-2, a specific small-molecule inhibitor of Hsp70/Bim complex, and Bcr-Abl knockdown to induce a panel of cancer cell lines apoptosis, we illustrated that Bcr-Abl could prevent cells from undergoing apoptosis mainly by driving the formation of Hsp70/Bim complex both in Bcr-Abl positive CML cells and ALL cells. Through cell-based Co-immunoprecipitation experiments, we identified that Bcr-Abl could stabilize oncogenic clients including AKT and eIF4E mainly by driving the formation of Hsp70/Bim complex in Bcr-Abl positive cells. Moreover, we identified that Bim mediates interactions of Hsp70 and Bak in Bcr-Abl positive cells. Together, the target identification of Hsp70/Bim complex could make it as a promising anticancer target for Bcr-Abl positive leukemia treatment.

A Novel ELISA-Based Peptide Biosensor Assay for Screening ABL1 Activity in vitro: A Challenge for Precision Therapy in BCR-ABL1 and BCR-ABL1 Like Leukemias.

The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (PABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The PABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (PABL-F) and fully-phosphorylated (PPHOSPHO-ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average PABL phosphorylation was significantly increased (basal vs. PABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p-value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to PPHOSPHO-ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on PABL; a lower signal was instead observed for NALM6 and REH (p < 0.001 and p < 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib (p < 0.001 for K562, NALM6, ALL-SIL and p < 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by PABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel PABL-ELISA assay on leukemic cells isolated from patient’s bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia (BCR-ABL1 positive) and a child affected by ALL (BCR-ABL1 negative) were performed. Phosphorylation of PABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the PABL-based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors.

The Rate of Cellular Energy Production of Muscle Cells Is Attenuated By Carnitine Intracellular Deficiency Caused By Imatinib Treatment

Introduction: Previous works identified that imatinib intake through the carnitine-specific OCTN2 (SLC22A5) transporter resulted in a significant decrease of carnitine intracellular concentrations in chronic myeloid leukemia (CML) and muscle cell lines. On contrary, even high doses of carnitine in preincubation did not influence imatinib cell intake capacity. Specifically performed inhibition of OCTN2 activity by vinorelbine resulted in block of carnitine cell intake, while imatinib intake was only slightly reduced (13-30%). This observation is in line with the knowledge that imatinib is transported also through other known SLC transporters. OCTN2 transporter is the major transporter for carnitine, an essential compound in cell energy metabolism. Presented work follows a hypothesis that non-equal competition between imatinib and carnitine intake through OCTN2 can lead to the carnitine intracellular deficiency, which can be in CML patients manifested by a disruption of skeletal muscle mitochondrial density and cause side effects like fatigue, muscle pain and cramps reported up to 80% of patients treated with imatinib (Kekale et al., 2015).Methods: Muscle cell HTB-153 (human rhabdomyosarcoma, ATCC HS 729), CML cell line KCL-22 (DSMZ ACC 519) were used for in vitro experiments. Intracellular concentration of imatinib, carnitine and metabolites were measured by chromatographic separation using XBridge Amide column (50x2.1mm, 3.5µm; Waters, Milford (MA), USA) and ZIC-pHILIC column (50x2.1mm, 5 µm; Merck, Darmstadt, Germany) coupled to tandem mass spectrometer (QTRAP 4000; Sciex, USA).Results: Carnitine, resp. L-carnitine transports long-chain fatty acids to mitochondria and its high rate is required especially in energetically demanding tissues such as skeletal and cardiac muscles. The concentrations of citric acid cycle (CAC) metabolites (citrate, malate, alpha-ketoglutarate, succinate, fumarate, 2-hydroxyglutarate, cis-aconitate), glycolysis (phosphoenolpyruvate, 3- phosphoglycerate, lactate), production of ATP, ADP and AMP were measured in HTB-153 cells 3 and 24 hours after imatinib treatment in vitro. The significant decrease of malate (CAC), lactate (glycolysis) and ATP levels were found at both time points after imatinib treatment compared to baseline. The same observations were found in KCL-22, which was used for comparison as BCR-ABL1 positive cell line. Additionally, significant decrease of succinate and 2-hydroxyglutarate (CAC) was detected in KCL-22 after imatinib treatment. Next, HTB-153 was incubated with imatinib (1-8 µM) for 24 hours and carnitine (8 µM) was supplied for last 3 hours of incubation, i.e., after 21 hours of imatinib treatment start. No significant changes were found in any metabolites of CAC and glycolysis. Production of ATP, ADP and AMP was not changed as well.Conclusions: Imatinib treatment of muscle (rhabdomyosarcoma) and CML cell lines caused a significant decrease of intracellular concentrations of carnitine. Significant decrease of ATP levels and of certain metabolites of CAC and glycolysis outlined that cells struggle from attenuated mitochondria energy production after imatinib treatment. This has not happened, if carnitine was supplied to the culture for final 3 hours of 24 hours incubation with imatinib. Observed data strongly support the hypothesis that decreased carnitine intake to the muscle cells due to competition with imatinib transport through OCTN2 attenuated mitochondria energy production. Interestingly, the clinical trial NCT03426722 (Chae H et al. 2019) showed that L-carnitine could effectively relieve imatinib-related muscle cramps and significantly increase QoL in patients with advanced gastrointestinal stromal tumor.Supported by GACR18-18407S, MZCR00023736 DisclosuresNo relevant conflicts of interest to declare.

Critical Interferon-Response Genes Are Induced By JAK2V617F but Repressed By BCR-ABL, and They Are Responsible for the Differential Effects of Interferon in PV Vs. CML

Interferon-alpha (IFNa) is successfully used as monotherapy in JAK2V617F positive myeloproliferative neoplasms (MPN) such as polycythemia vera (PV), but with only limited success in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa in JAK2V617F vs. BCR-ABL positive cells.In 32D cells stably transduced with JAK2V617F, IFNa alone (100 U/mL) significantly decreased the viability to less than 50% of control but had no significant effect on 32D-BCR-ABL cell viability, as assessed by MTT assays. Gene expression microarray data of 32D-JAK2V617F cells revealed strong upregulation of a number of interferon-stimulated genes compared to empty vector-transduced (EV) cells, and RT-PCR validation confirmed significantly increased expression of Stat1, Stat2 and Irf9 and reversal of these effects by ruxolitinib treatment. Conversely, BCR-ABL led to a decrease of these three genes, and this was reversed by imatinib. Similar responses were seen in human HEL and K562 cells. Histone (H3K9) acetylation of Stat1, Stat2 and Irf9 promoters was significantly higher in 32D-JAK2V617F vs. 32-BCR-ABL cells, correlating with gene expression. IFNa treatment increased Stat1, Stat2 and Irf9 mRNA in both 32D-JAK2V617F and 32D-BCR-ABL cells, and this effect was reduced by ruxolitinib but potentiated by imatinib. In line with these results, the phosphorylation of STAT1 (P-STAT1) was induced by IFNa in all cell lines. In addition, Stat1, Stat2 and Irf9 mRNA was significantly downregulated in peripheral blood samples from CML but not PV patients, as compared to healthy controls (all subjects provided written informed consent, as approved by the local ethics committee). Likewise, Stat1 and Stat2 expression was significantly downregulated in SCLtTA/BCR-ABL but not JAK2V617F transgenic mice.We next analyzed whether ectopic expression of STAT1 or STAT2 or their dominant-negative Y/F mutants affected the IFNa sensitivity of 32D-JAK2V617F vs. 32D-BCR-ABL cells. While there was little apoptosis in empty-vector- or STAT1-overexpressing 32D-BCR-ABL cells, overexpression of the STAT1Y701F mutant in these cells showed a significantly increased basal level of apoptosis, which was further enhanced by IFNa (p<0.05). This was associated with IFNa-induced P-STAT1 but complete absence of P-STAT2. Conversely, EV- or STAT1-overexpressing 32D-JAK2V617F cells showed strong IFNa-induced apoptosis, while STAT1Y701F overexpression in these cells led to abrogation of IFNa-induced apoptosis. This was associated with decreased P-STAT1 but preserved P-STAT2. On the other hand, STAT2 overexpression decreased P-STAT1 and P-STAT2 and led to mild IFNa-induced apoptosis in 32D-BCR-ABL cells, but preserved the high P-STAT1 and P-STAT2 as well as potent IFNa responsiveness in 32D-JAK2V617F cells.In conclusion, JAK2V617F and BCR-ABL induce opposite effects on IFNa target genes in vitro and in vivo, suggesting that JAK2V617F but not BCR-ABL sensitizes cells to interferon. Moreover, while the major factor of IFNa-induced apoptosis in JAK2V617F positive cells is an increase of P-STAT1, the major determinant in BCR-ABL positive cells is a decrease of P-STAT2. Together, these results may possibly explain the differences of clinical responses to IFNa monotherapy in PV vs. CML. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Koschmieder:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche: Other: Clinical Trial participation.

Allosteric Bcr-Abl Inhibitor ABL001 Overcomes Bcr-Abl Tyrosine Kinase Inhibitor Resistance and Enhances MDM2 Inhibitor CGM097 Activity in Blast Crisis CML Cells

Despite Bcr-Abl tyrosine kinase inhibitors (TKIs) are highly effective in CML, development of TKI-resistance leading to blast crisis (BC) progression is still a challenge in CML therapy. This has motivated the generation of novel agents and combination strategies to overcome the resistance. ABL001 is a small molecule allosteric Bcr-Abl kinase inhibitor that occupies the myristoyl pocket, leading to a kinase autoinhibition conformation. ABL001 has entered phase I clinical trials to treat patients with chronic, accelerated, or blast phase CML that relapsed from TKIs (Hughes et al., ASH 2016). Its activity in BC CML and ability to enhance the traditional TKIs that target ATP binding site are under investigation. TP53, a key tumor suppressor gene plays a crucial role in mediating cell death in part through regulating the expression or activity of Bcl-2 family proteins. Although highly mutated in solid tumors, the mutation rate of TP53 is relatively low in CML. However, p53 antagonist, MDM2 can be regulated by Bcr-Abl signaling and ARF, which inhibits MDM2 is frequently deleted in BC CML suggesting therapeutic potentials of inhibition of MDM2 in BC CML. Indeed, activation of p53 by MDM2 inhibition has been shown to enhance the toxicity of TKIs in BC CML (Carter et al., Oncotarget 2015, 6(31):30487). In this study, we examined the activity of ABL001 in CML cells and investigated if ABL001 enhances the activity of TKIs and synergizes with MDM2 inhibitor CGM097 in samples from heavily treated TKI resistant BC CML patients.BaF3 cells expressing BCR-ABL with or without mutations, human CML cell lines (KBM5 and KBM5-STI-T315I), and peripheral blood mononuclear cells (PBMC) isolated from primary BC CML patient samples were treated with various doses of ABL001, CGM097 or Nilotinib alone or in combination. Apoptosis was determined by Annexin V/7-AAD staining in cell lines and CD45+ (bulk), CD34+CD38+ and CD34+CD38- cells of CML patient PBMC samples. EC50 was calculated using CompuSyn or Graphpad. Protein levels were examined by Western blotting.We found that ABL001 induced apoptosis in cell lines expressing BCR-ABL (BaF3p210 and KBM5) or mutant BCR-ABL (BaF3p210E255K and KBM5-STI-T315I) but had limited activity in BaF3p210T315I cells. Importantly, ABL001 induced apoptosis in bulk, CD34+CD38+, or/and CD34+CD38- cell populations at low nM concentrations in 5 out of 6 BC CML patient samples tested. Patient samples that responded to ABL001 had either T315I and/or E255 mutations in BCR-ABL kinase domain. ABL001 inhibited Bcr-Abl signaling as measured by the levels of p-Bcr-Abl/p-Stat5/p-Crkl in BCR-ABL positive cells with or without BCR-ABL mutations. ABL001 showed higher activity than Nilotinib in 4 out of 5 responding patient samples. Nilotinib did not significantly enhance ABL001-induced apoptosis in the tested BC CML patient samples and CML cell lines. Inhibition of MDM2 with CGM097 activated p53 and induced apoptosis in BC CML patient samples. The activity of ABL001 was enhanced by CGM097 in the majority of patient samples which did not have p53 mutations. ABL001 combined with CGM097 was more effective than the combination of nilotinib with CGM097. The combinations of TKI and CGM097 were more effective in killing leukemia cells than each agent alone even when leukemia cells were co-cultured with MSCs. Neither ABL001 or CGM097 alone, nor the combination showed toxicity on normal bone marrow stem/progenitor cells.In conclusion, our results demonstrate that the allosteric Bcr-Abl Inhibitor ABL001 overcomes TKI-resistance and enhances MDM2 inhibitor CGM097 activity in BC CML cells. The combination of ABL001 and CGM097 warrants clinical evaluation. DisclosuresCarter:novartis: Research Funding; Daiichi Sankyo: Research Funding.

Challenging the Prognostic Value of MRD: Sequential Tumor-Ablative Conditioning of FA5-Bucy for Ph-Positive Acute Lymphoblastic Leukemia

Background:Minimal residual disease (MRD) quantification prior to allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a powerful predictor for post-transplant outcome. Thus, new strategies with modified transplantation procedures are needed to reverse the outcomes of that remain MRD-positive despite multiple induction attempts.OBJECTIVE:To determine the prognostic relevance of MRD status in patients with acute lymphoblastic leukemia (ALL), especially those with Philadelphia chromosome-positive ALL (Ph+ ALL), receiving the conditioning regimen of FA5-Bucy, registered on http:// ClinicalTrials.gov (NCT02328950).METHODS:We retrospectively reviewed our HSCT experience using FA5-Bucy, i.e., 5-day salvage chemotherapy (Fludarabine/Ara-C) and conditioning (Busulfan/ Cyclophosphamide) for patients with ALL. A total of 48 consecutive patients from 2013 to 2016 are analyzed. 11 out of 48 (7 males and 4 females) were diagnosed with Ph+ ALL with the induction treatments including a tyrosine kinase inhibitor (TKI). Median age was 30 years (range, 8-52 years). Among whom four (36.4%) achieved complete remission(CR)at transplantation, Six (54.5%) with detectable residual P190 BCR-ABL positive leukaemic cells, while 1 (9.1%) not in the status of remission with 39.5% blasts in the bone marrow (BM). The other 37 cases were Ph-negative (Ph-) ALL, with the median age of 21 years (range, 2-61 years). Among whom 25 (67.6%) patients were in CR, 6 (16.2%) MRD-positive and 6 (16.2%) with 23.5%-98% BM blasts. The MRD status was monitored by four-color FCM in Ph- group, and by RT-PCR to detect the chimeric bcr-abl mRNA transcript in Ph+ group. Patients were transplanted either from an HLA-identical sibling or haplo-identical related donors. The GVHD prophylaxis consisted of ATG, CsA, MMF and short-term MTX. None of these patients had severe infection or organ failure before HSCT.RESULTS:All patients achieved successful engraftment and full donor chimerism. Patients with Ph+ ALL seems have a better outcome than that with Ph- ALL (3-year OS of 54.5% vs 37.8%, P=0.245). With a median follow-up of 666 (83-1276) days, the 1y-OS and 1y-DFS were 72.7%, 3y-OS and 3y-DFS were 54.5% in Ph+ group, respectively, and non-relapse mortality and relapse incidence were 18.2% and 10%. MRD monitoring is an important tool for relapse prediction in Ph- ALL patients (3-year OS and DFS of MRD-positive vs MRD-negative =16.7% vs 56%, P=0.021).However, the profile of OS and DFS was similar in Ph+ patients with bcr-abl positive to that with bcr-abl negative (3-year OS and DFS of bcr-abl positive vs bcr-abl negative= 57.1% vs 50%, P=0.744). Severe acute-GvHD (aGVHD) occurred in 3/11 patients. Few patients experienced mild-to-moderate toxicity, and main causes of death were aGVHD.CONCLUSION:We concluded that allo-HSCT with the sequential tumor-ablative conditioning of FA5-Bucy offers great benefits for the patients with Ph+ ALL, with the potentially capability of reducing or overcoming the negative impacts of MRD. Thus creates exciting transplant opportunities for patients not in remission to be cured. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Assessment of Standardization and International Scale (IS) Alignment Efforts of BCR-ABL1 Testing By the Chinese CML Alliance in an International External Quality Assessment Program

Sequential BCR-ABL1 mRNA level measurement by reverse transcription quantitative polymerase chain reaction (RT-qPCR) is embedded in management of chronic myeloid leukaemia (CML) patients.United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) has been providing External Quality Assessment (EQA)/Proficiency Testing (PT) to 200 laboratories undertaking BCR-ABL1 RT-qPCR analysis since 2007.The Chinese CML Alliance (CCA), established in 2011 for the purpose of the standardization of BCR-ABL1 measurement includes 58 laboratories to date. Peking University People's Hospital (PUPH), the Chinese regional reference centre, has acquired conversion factors (CFs) for reporting BCR-ABLIS through sample exchanges with the Adelaide IMVS reference laboratory. Other members acquired their laboratory specific CFs thorough sample exchange with PUPH, and their CFs were regularly revalidated through testing distributed samples by PUPH.We reviewed performance of CCA laboratories in UK NEQAS LI's BCR-ABL1 Quantification program to evaluate if technical standardization initiatives and local result alignment to the International Scale (IS) (defined as BCR-ABLIS) increased accuracy and precision of BCR-ABL1 measurement by CCA labs.MethodsThis study was facilitated by Diaceutics and Novartis. 5 CCA laboratories were included in 3 EQA/PT send-outs provided by UK NEQAS LI. Six standard samples and one additional educational sample were tested by CCA laboratories, alongside up to 207 other international participants. Samples comprised two lyophilized cell line mixtures containing dilutions of BCR-ABL1 (e14a2) positive K562 cells in a background of BCR-ABL1 negative HL60 cells.For each send-out, CCA laboratories were assessed against the IS robust mean BCR-ABLIS for each sample tested and their proximity to the robust mean log reduction between two standard trial samples tested, providing information on CCA laboratory IS alignment and assay linearity.ResultMedian BCR-ABLIS sample levels in this study (calculated from all participant data) ranged from 6.1% to 0.002% (Figure 1). All five CCA laboratories used in-house based methods to measure BCR-ABL1; 3 based their in-house method on the Europe Against Cancer protocols. ABL1 was detected in all samples by the 5 centres with 4 achieving ABL1 levels >100,000. Additionally, 4/5 detected BCR-ABL1 transcript in all seven samples but 1/5 laboratories failed to detect any transcript in 1 sample (the sample that had the lowest level of transcript (median BCR-ABLIS = 0.002%; ~MR 4.7)).4/5 laboratories measured BCR-ABL1IS levels within +2 SD of the robust means for individual sample and log reduction analysis undertaken between samples for each send-out.DiscussionThis study shows that efforts by the CCA to standardize BCR-ABL1 measurement and align local results to the IS have been successful. Satisfactory results (within +2SD of the robust mean) were obtained in 4/5 laboratories providing reassurance that patient testing in these laboratories is accurate and precise. A root cause analysis undertaken in the laboratory with results >+2SD from the robust mean found a pre-analytical sample transposition error that when reanalyzed was within the acceptable range for both individual sample and log reduction results.The study showed that some CCA laboratories omitted the BCR-ABL1 standard curve, something not recommended in technical guidelines (Foroni et al., 2011) as erroneous results may occur due to unrecoqnized assay drift or suboptimal amplification. Standard curves for both ABL1 and BCR-ABL1 should always be run; however it is acceptable to run a standard curve each day for multiple runs but these must use the same master mix used for the sample quantification (Foroni et al., 2011).Future studies will include more laboratories in the CCA network to assess if the use of internationally recognized calibrants can achieve even greater standardization.Figure 1. All participant BCR-ABLIS results for the 7 samples tested. Blue markers = non CCA laboratories; red marker = CCA laboratories. Long horizontal black lines = IS median. Short horizontal black lines = range.*Ya-Zhen Qin andStuart Scott contributed equally to this study. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Relationship between Oxidative Stress and Imatinib Resistance in Model Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) develops due to the presence of the BCR-ABL1 protein, a target of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), used in a CML therapy. CML eradication is a challenge due to developing resistance to TKIs. BCR-ABL1 induces endogenous oxidative stress leading to genomic instability and development of TKI resistance. Model CML cells susceptible or resistant to IM, as well as wild-type, non-cancer cells without the BCR-ABL1 protein were treated with IM, hydrogen peroxide (H2O2) as a model trigger of external oxidative stress, or with IM+H2O2. Accumulation of reactive oxygen species (ROS), DNA damage, activity of selected antioxidant enzymes and glutathione (GSH), and mitochondrial potential (MMP) were assessed. We observed increase in ROS accumulation in BCR-ABL1 positive cells and distinct levels of ROS accumulation in IM-susceptible cells when compared to IM-resistant ones, as well as increased DNA damage caused by IM action in sensitive cells. Depletion of GSH levels and a decreased activity of glutathione peroxidase (GPx) in the presence of IM was higher in the cells susceptible to IM. IM-resistant cells showed an increase of catalase activity and a depletion of MMP. BCR-ABL1 kinase alters ROS metabolism, and IM resistance is accompanied by the changes in activity of GPx, catalase, and alterations in MMP.

Expression of inducible NOS is indispensable for the antiproliferative and proapoptotic effect of imatinib in BCR-ABL positive cells.

Chronic myeloid leukemia (CML) is characterized by constitutive BCR-ABL kinase activity, an aggressive proliferation of immature cells, and reduced differentiation. Targeting tyrosine kinase activity of BCR-ABL with imatinib is an effective therapy for the newly diagnosed CML patients; however, 20%-30% of the patients initially treated with imatinib eventually experience treatment failure. Therefore, early identification of these patients is of high clinical relevance. In the present study, we by undertaking a direct comparison of inducible NOS (iNOS) status in neutrophils from healthy volunteers, newly diagnosed, imatinib responder, and resistant CML patients as well as by conducting in vitro studies in K562 cells demonstrated that inhibition of BCR-ABL by imatinib or siRNA significantly enhanced NO generation and iNOS expression. Indeed, patients exhibiting treatment failure or imatinib resistance were less likely to induce NO generation/iNOS expression. Our findings further demonstrated that imatinib mediated antiproliferative and proapoptotic effect in BCR-ABL+ cells associated with enhanced iNOS expression, and it was significantly prevented in the presence of L-NAME, 1400W, or iNOS siRNA. Overexpression of iNOS in K562 cells expectedly enhanced imatinib sensitivity on cytostasis and apoptosis, even at lower concentration (0.1μM) of imatinib. Mechanistically, imatinib or BCR-ABL siRNA following deglutathionylation of NF-κB, enhanced its binding to iNOS promoter and induced iNOS transcription. Deglutathionylation of procaspase-3 however associated with increased caspase-3 activity and cell apoptosis. Taken together, results obtained suggest that monitoring NO/iNOS level could be useful to identify patients likely to be responsive or resistant to imatinib and can be used to personalized alternative therapy.

Synthesis, molecular modeling, and biological evaluation of novel imatinib derivatives as anticancer agents.

Different derivatives of imatinib were synthesized by a 3-step reaction method. The structures of the new compounds were characterized by spectroscopic methods. For quantitative evaluation of the biological activity of the compounds, MTT assays were performed, where four BCR-ABL negative leukemic cell lines (Jurkat, Reh, Nalm-6 and Molt-4), one BCR-ABL positive cell line (K562), and one non-leukemic cell line (Hek293T) were incubated with various concentrations of the derivatives. Although imatinib was specifically designed for the BCR-ABL protein, our results showed that it was also effective on BCR-ABL negative cell lines except for Reh cell line. Compound 9 showed lowest IC50 values against Nalm-6 cells as 1.639 μM, also the values of Compound 10 for each cell were very close to imatinib. Molecular docking simulations suggest that except for compound 6, the compounds prefer a DFG-out conformation of the ABL kinase domain. Among them, compound 10 has the highest affinity for ABL kinase domain that is close to the affinity of imatinib. The common rings between compound 10 and imatinib adopt exactly the same conformation and same type of interactions in the ATP binding site with the ABL kinase domain.

Design, synthesis, and biological evaluations of novel 3-amino-4-ethynyl indazole derivatives as Bcr-Abl kinase inhibitors with potent cellular antileukemic activity.

Breakpoint cluster region-Abelson (Bcr-Abl) kinase is a key driver in the pathophysiology of chronic myelogenous leukemia (CML). Broadening the chemical diversity of Bcr-Abl kinase inhibitors with novel chemical entities possessing favorable target potency and cellular efficacy is a current medical demand for CML treatment. In this respect, a new series of ethynyl bearing 3-aminoindazole based Bcr-Abl inhibitors has been designed, synthesized, and biologically evaluated. The target compounds were designed based on introducing the key structural features of ponatinib, alkyne spacer and diarylamide, into the previously reported indazole II to improve its Bcr-Abl inhibitory activity and overcome its poor cellular potency. All target compounds elicited potent activity against Bcr-AblWT with sub-micromolar IC50 values ranging 4.6-667nM. In addition, certain derivatives exhibited promising potency over the clinically imatinib-resistant Bcr-AblT315I. Among the target molecules, compounds 9c, 9h and 10c stood as the most potent derivatives with IC50 values of 15.4nM, 4.6nM, and 25.8nM, respectively, against Bcr-AblWT. Interestingly, 9h showed 2 folds and 3.6 times superior potency to the lead indazole II and 10c, respectively, against Bcr-AblT315I. Molecular docking of 9h pointed out its possibility to be a type II kinase inhibitor. Furthermore, all compounds, except 9b, showed highly potent antiproliferative activity against the Bcr-Abl positive leukemia K562cell (MTT assay) surpassing the modest activity of lead indazole II. Moreover, the most potent members 9h and 10c exerted potent antileukemic activity against NCI leukemia panel, particularly K562cell (SRB assay) with GI50 less than 10nM, being superior to the FDA approved drug imatinib. Further biochemical hERG and cellular toxicity, phosphorylation assay, and NanoBRET target engagement of 9h underscored its merits as a promising candidate for CML therapy.

RUNX1 mutations in blast-phase chronic myeloid leukemia associate with distinct phenotypes, transcriptional profiles, and drug responses

Blast-phase chronic myeloid leukemia (BP-CML) is associated with additional chromosomal aberrations, RUNX1 mutations being one of the most common. Tyrosine kinase inhibitor therapy has only limited efficacy in BP-CML, and characterization of more defined molecular subtypes is warranted in order to design better treatment modalities for this poor prognosis patient group. Using whole-exome and RNA sequencing we demonstrate that PHF6 and BCORL1 mutations, IKZF1 deletions, and AID/RAG-mediated rearrangements are enriched in RUNX1mut BP-CML leading to typical mutational signature. On transcriptional level interferon and TNF signaling were deregulated in primary RUNX1mut CML cells and stem cell and B-lymphoid factors upregulated giving a rise to distinct phenotype. This was accompanied with the sensitivity of RUNX1mut blasts to CD19-CAR T cells in ex vivo assays. High-throughput drug sensitivity and resistance testing revealed leukemia cells from RUNX1mut patients to be highly responsive for mTOR-, BCL2-, and VEGFR inhibitors and glucocorticoids. These findings were further investigated and confirmed in CRISPR/Cas9-edited homozygous RUNX1−/− and heterozygous RUNX1−/mut BCR-ABL positive cell lines. Overall, our study provides insights into the pathogenic role of RUNX1 mutations and highlights personalized targeted therapy and CAR T-cell immunotherapy as potentially promising strategies for treating RUNX1mut BP-CML patients.