Chronic Myeloid Leukemia Therapy Research Articles

A Quality by Design (QbD) driven gradient high performance liquid chromatography method development for the simultaneous estimation of dasatinib and nilotinib in lipid nanocarriers

Tyrosine kinase inhibitors (TKIs) are effective as a targeted treatment for chronic myeloid leukemia (CML), which can selectively suppress BCR-ABL1 kinase activity. CML therapy with TKIs combination has been supported by in-vitro, in-vivo, and patient-based data where the nilotinib-dasatinib co-administration has exerted superior anticancer efficacy with greater cellular uptake, less resistance to chemotherapy, and no additive adverse events encountered. Therefore, it is essential to develop a suitable analytical method for the simultaneous estimation of these drugs in the developed novel lipid nanocarriers like liposomes. Design of Experiment (DoE) has been implemented as a tool of QbD to systematically investigate the relation between the HPLC method attributes and analytical responses, i.e., chromatographic detection, quantification, and peak properties for dasatinib and nilotinib. An Ishikawa diagram is constructed to delineate possible influencing variables to the analytical performances. Afterward, 4 factors 2 level full factorial design (FFD) was employed to model and identify the main effects and interaction effects between the factors selected after the initial risk assessment. The suggested design space for optimized chromatographic conditions by QbD analysis is linear within the selected range of drug concentrations, accurate and precise, sensitive, and robust according to the ICH guidelines. The optimal method is comprised of a 1 mL/min flow rate of mobile phase (ACN and 20 mM KH2PO4 of pH 7.00) in gradient mode at 25 °C column temperature for 20 μL sample injection volume and detection wavelength fixed at 297 nm. Most importantly, this novel HPLC method is simple and selective enough to evaluate dasatinib and nilotinib content in the lipid nanocarriers.

Disparities in frontline treatment and overall survival in the era of targeted tyrosine kinase inhibitor therapy for chronic myeloid leukemia: 2004-2021.

11187 Background: The treatment of chronic myeloid leukemia (CML) was revolutionized in 2001 with the introduction of the BCR::ABL tyrosine kinase inhibitor (TKI), imatinib. In 2006, dasatinib was approved, followed by nilotinib in 2007. Two other TKIs, bosutinib & ponatinib were approved in 2012, followed by the allosteric ABL myristoyl inhibitor asciminib in 2021, providing multiple options for patients (pts) who have refractory disease. In this study we examine how survival has improved in CML pts with the availability of multiple TKI’s and identify disadvantaged socioeconomic & demographic groups which continue to have disparate survival. Methods: The National Cancer Database was used to identify CML pts diagnosed (dx) from 2004-2021. Demographic, treatment, & overall survival (OS) were compared by era of TKI availability, with pts dx from 2004-2005 considered to be treated in the imatinib era, 2006-2011 for dasatinib & nilotinib, 2012-2020 with access to bosutinib & ponatinib, and 2021 for asciminib. Results: Of 44,993 CML pts identified, 55.8% were male & 82.5% were White, with median age of 58 years. The portion of pts who were untreated (due to contraindication or early death) decreased by each time period, from 17.8% in the imatinib era to only 7.8% in 2021. The rate of upfront transplant decreased from 2.5% from 2004-2005, to 0.8% in 2021. For dx 2004-2005 median OS was 11.5 yrs (95% CI 10.6-12.5). This increased to 13.6 yrs (CI 13.1-14.1), with age-adjusted hazard ratio (HR) of 0.86 (CI 0.81-0.91) for pts dx 2006-2011, with median OS unreached for 2012-2020, HR 0.67 (CI 0.63-0.71, p<0.001); referenced to 2004-2006. Survival at 1-, 5- & 10-yrs was improved for each subsequent time period of dx, with 5-yr OS of 65%, 71%, & 76%, respectively, p<0.001. On multivariate cox regression adjusted for yr of dx, features associated with reduced OS included age (HR 1.06 [95% CI 1.05-1.06], p<0.001 for each yr), Black race (HR 1.11 [95% CI 1.05-1.18], p<0.001), increased comorbidity index (HR 1.94 [95% CI 1.84-2.04], p<0.001 for index ≥2), uninsured (HR 2.20 [95% CI 2.01-2.41], p<0.001), or insured through Medicaid (HR 2.40 [95% CI 2.23-2.58], p<0.001). Conclusions: Survival for CML pts has significantly improved during the last 20 yrs with the availability of each additional TKI, likely related to increased treatment options for pts with resistance or intolerance. Additional TKI options, particularly approval of ponatinib for patients with T315I mutation, have a secondary benefit of decreasing the utilization of allogenic transplant. Though survival has improved for all pts, pts from traditionally underserved populations, including pts who are underinsured or from racial minority groups, have reduced OS. Continued advances in treatment & efforts to improve access to care for underserved populations is vital to achieve equitable survival outcomes in CML.

Advancements and Future Prospects in Molecular Targeted and siRNA Therapies for Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is an oncological myeloproliferative disorder that accounts for 15 to 20% of all adult leukemia cases. The molecular basis of this disease lies in the formation of a chimeric oncogene BCR-ABL1. The protein product of this gene, p210 BCR-ABL1, exhibits abnormally high constitutive tyrosine kinase activity. Over recent decades, several targeted tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1 have been developed and introduced into clinical practice. These inhibitors suppress BCR-ABL1 activity through various mechanisms. Furthermore, the advent of RNA interference technology has enabled the highly specific inhibition of BCR-ABL1 transcript expression using small interfering RNA (siRNA). This experimental evidence opens avenues for the development of a novel therapeutic strategy for CML, termed siRNA therapy. The review delves into molecular genetic mechanisms underlying the pathogenesis of CML, challenges in CML therapy, potential molecular targets for drug development, and the latest results from the application of siRNAs in in vitro and in vivo CML models.

Results of ponatinib as frontline therapy for chronic myeloid leukemia in chronic phase.

Ponatinib is a third-generation BCR::ABL1 tyrosine kinase inhibitor (TKI) with robust activity in Philadelphia chromosome-positive leukemias. Herein, we report the long-term follow-up of the phase 2 trial of ponatinib in chronic myeloid leukemia in chronic phase. Patients received ponatinib 30 to 45 mg/day. The primary end point was the rate of 6-month complete cytogenetic response (CCyR). The study was held in June 2014 because of the risk of cardiovascular toxicity, requiring patients to change TKI. Fifty-one patients were treated with ponatinib (median dose, 45 mg/day). Median age was 48 years (range, 21-75); 30 (59%) had baseline cardiovascular comorbidities. Median treatment duration was 13 months (range, 2-25). Fourteen patients (28%) discontinued ponatinib because of toxicities, 36 (71%) after the Food and Drug Administration warning/study closure, and one for noncompliance. Dasatinib was the most frequently chosen second-line TKI (n=34; 66%). Among 46 patients evaluable at 6 months, 44 (96%) achieved CCyR, 37 (80%) major molecular response, 28 (61%) MR4, and 21 (46%) MR4.5. The cumulative 6-month rates of CCyR, major molecular response, MR4, and MR4.5 were 96%, 78%, 50%, and 36%, respectively. Durable MR4 ≥24 or ≥60 months was observed in 67% and 51% of patients, respectively. The 24-month event-free survival rate was 97%. After a median follow-up of 128 months, the 10-year overall survival rate was 90%. Eight patients (16%) had serious grade 2 to 3 cardiovascular adverse events, leading to permanent discontinuation in five (10%). Ponatinib yielded high cytogenetic and molecular responses in newly diagnosed chronic myeloid leukemia in chronic phase. Its use in the frontline setting is hindered by arterio-/vaso-occlusive and other severe toxicities.

Disparities in Frontline Treatment and Overall Survival in the Era of Targeted Tyrosine Kinase Inhibitor Therapy for Chronic Myeloid Leukemia: 2004-2021

Disparities in Frontline Treatment and Overall Survival in the Era of Targeted Tyrosine Kinase Inhibitor Therapy for Chronic Myeloid Leukemia: 2004-2021

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

BackgroundDespite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.MethodsTranscriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.ResultsTranscriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.ConclusionsOverall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Abstract 7469: Inflammation plays an important role in the progression of chronic myeloid leukemia

Abstract The development of Tyrosine Kinase Inhibitors (TKIs) resulted in a significant increase in the life expectancy of patients with chronic myeloid leukemia (CML). Nevertheless, TKIs still fail in a significant proportion of therapy-naïve patients, or their effectiveness is hampered by secondary resistance. It is therefore urgent to explore the underlying mechanisms in order to develop new therapeutic approaches to cure CML instead of controlling the disease with a lifelong TKI therapy. Using the ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML we show that BCR::ABL1 leads to the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression. This is supported by our data from CML patients, where we also observe higher levels of pro-inflammatory cytokines in the serum compared to healthy individuals. Furthermore, we demonstrate that BCR::ABL1 drives the expansion of bone marrow derived mast cells (BMMCs) and sensitizes them for FcεRI triggered degranulation. Interestingly, these BMMCs show differences in their lipid metabolism. Hence, sphingomyelin and lysophosphatidylcholine, both known to be involved in inflammation and cancer progression, are upregulated in BCR::ABL1-positive mast cells compared to their negative counterparts. By crossing in the Cpa3Cre mediated model for mast cell deficiency, we demonstrate for the first time that the lack of mast cells prevents the BCR::ABL1 induced upregulation of pro-inflammatory cytokines and also the development of splenomegaly. In addition, we were able to show that splenomegaly in CML patients is associated with high bone marrow mast cell counts and that an upregulation of pro-inflammatory cytokines in patient serum samples correlated with tryptase levels. In summary, our study identifies mast cells and their inflammatory cytokines as important factors in disease progression and suggests considering them as additional targets in CML therapy. Citation Format: Melanie Langhammer, Julia Schöpf, Timo Jaquet, Katharina Horn, Moritz Angel, Daniel Mohl, Simon Lagies, Corinna Spohr, Daniel Christen, Franziska Maria Uhl, Tiago Maié, Henrike Jacobi, Thorsten B. Feyerabend, Julia Huber, Marcus Panning, Cassian Sitaru, Ivan Costa, Robert Zeiser, Konrad Aumann, Heiko Becker, Till Braunschweig, Steffen Koschmieder, Bernd Kammerer, Khalid Shoumariyeh, Michael Huber, Mirle Schemionek-Reinders, Tilman Brummer, Sebastian Halbach. Inflammation plays an important role in the progression of chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7469.

The potential of circHIPK3 as a biomarker in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by leukocytosis and left shift. The primary molecular alteration is the BCR::ABL1, chimeric oncoprotein with tyrosine kinase activity, responsible for the initial oncogenesis of the disease. Therapy of CML was revolutionized with the advent of tyrosine kinase inhibitors, but it is still not considered curative and may present resistance and serious adverse effects. Discoveries in CML inaugurated a new era in cancer treatment and despite all the advances, a new biomarker is needed to detect resistance and adverse effects. Circular RNAs (circRNAs) are a special type of non-coding RNA formed through a process called backsplicing. The majority of circRNAs are derived from protein-coding genes. CircHIPK3 is formed from the second exon of the HIPK3 gene and has been found in various pathologies, including different types of cancer. New approaches have demonstrated the potential of circular RNAs in cancer research, and circHIPK3 has shown promising results. It is often associated with cellular regulatory pathways, suggesting an important role in the molecular dynamics of tumors. The identification of biomarkers is an important tool for therapeutic improvement; thus we review the role of circHIPK3 and its potential as a biomarker in CML.

Drug-induced chylothorax as a rare pleural complication in dasatinib therapy for chronic myeloid leukaemia.

Chylothorax is a lymphatic chylous pleural effusion typically associated with traumatic (iatrogenic, non-iatrogenic) and non-traumatic (infections, malignancy, lymphatic disorders) aetiologies. Drug-induced chylothorax is uncommon and mostly reported in association with BCR-ABL tyrosine kinase inhibitor therapy.

High Efficacy and Safety of Asciminib in a Chronic Myeloid Leukemia Patient with Chronic Kidney Disease Following Renal Transplantation.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm driven by the BCR::ABL1 tyrosine kinase. Tyrosine kinase inhibitors (TKIs) have been established as standard therapies for CML. However, some CML patients experience TKI intolerance. Asciminib was approved for CML patients either intolerant or refractory to TKI therapy. We herein report a 63-year-old CML patient who underwent renal transplantation and exhibited TKI intolerance. He was switched to asciminib, which achieved a deep molecular response without exacerbation of the renal function. Our experience revealed that asciminib is effective and safe for CML patients complicated with chronic kidney disease.

KIR-генетические факторы и ответ на терапию ингибиторами тирозинкиназ при хроническом миелоидном лейкозе

The development of tyrosine kinase inhibitors (TKIs) and their introduction into clinical practice considerably improved the prognosis for chronic myeloid leukemia (CML) patients. About 50 % of patients with achieved deep molecular response are eligible for safe TKI discontinuation. Despite these advances, no reliable biomarkers are known to predict a response and sustaining treatment-free remission after TKI withdrawal. As TKIs do not destroy leukemic stem cells, which can be responsible for relapse, critical importance in CML is attached to natural killers (NK-cells) having antitumor activity. Functional activity of NK-cells is evaluated by expression level and repertoire of killer cell immunoglobulin-like receptors (KIR). Current studies demonstrate that a patient’s KIR genotype affects the probability of achieving early and deep molecular responses to first- and second-generation TKIs, progression-free and overall survivals, and sustaining treatment-free remission. On that ground, KIR-genetic factors can be regarded as promising predictors of response to TKI therapy in CML. Early clinical studies, which dealt with monoclonal antibodies blocking the inhibitory KIR in order to increase NK-cell activity, revealed an acceptable safety profile and efficacy in some hematological diseases (such as acute myeloid leukemia, multiple myeloma, Т-cell lymphoma) if used in combination with cytostatic drugs or antitumor monoclonal antibodies. KIR genotype determination can contribute to the development of effective therapies of this malignant hematological tumor.

Comorbidities and outcomes of patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors: a real-world, nationwide, retrospective study from Hungary.

Purpose: This study aimed to provide real-world evidence on the characteristics, treatment patterns, and outcomes of patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitor (TKI) treatment in Hungary between 2011 and 2019. Patients and methods: This nationwide, retrospective study included patients who were newly diagnosed with CML in Hungarian clinical practice between January 2011 and December 2019. The analysis was based on the reimbursed prescription claims for imatinib, bosutinib, dasatinib, nilotinib, or ponatinib with the ICD-10 code C9210 in a public pharmacy between January 2009 and December 2019 using data from the National Health Insurance Fund (NHIF) of Hungary. CML incidence and prevalence, TKI treatment patterns, comorbidities, and overall survival (OS) were examined. Results: Between 2011 and 2019, altogether 1,407 patients were diagnosed with CML, with an annual average of 156 patients. The number of patients newly initiating first-line TKI therapy for CML significantly increased between 2011 and 2019 (2011: n = 136 vs. 2019: n = 191; p = 0.0043). Nilotinib was typically prescribed for younger patients (≤64years), while older patients (≥65years) mostly received imatinib. The most common comorbidity of CML patients was hypertension, and the proportion of patients with other malignancies was relatively high in all treatment groups. 5-year OS was 77.1% during the whole study period. Patients initiating first-line TKI treatment for CML in 2015 had significantly better 4-year OS compared to those starting treatment in 2011 (82.4% vs. 73.5%, respectively, (HR 0.53 (95%CI 0.32-0.87) p = 0.0118). Conclusion: This study is the first to provide insights into the characteristics, treatment patterns, and outcomes of CML patients treated with TKIs in Hungarian clinical practice between 2011 and 2019. We found slightly lower OS rates compared to other European countries, however, there was a statistically significant improvement in 4-year OS during the study period. The management of CML was in line with international guidelines and recommendations.

Exploring the binding mechanism of a small molecular Hsp70-Bim PPI inhibitor through molecular dynamic simulation.

The interface of Hsp70-Bim protein-protein interaction (PPI) has been identified as a specific target for Chronic Myeloid Leukemia (CML) therapy and the specific inhibitors were developed to exhibit in vivo anti-leukemia activities. Herein, we explored the binding mechanism of a Hsp70-Bim inhibitor, 6-(cyclohexylthio)-3-((2-morpholinoethyl) amino)-1-oxo-1H-phenalene-2-carbonitrile (S1g-6), to Hsp70 at the atomic level by MD simulation. TYR-149, THR-222, ALA-223, and GLY-224 on Hsp70 were identified as four key residues that contribute to Hsp70/S1g-6 complex. Moreover, the site mutation validation demonstrated the TYR-149 of Hsp70 is a "hot-spot" in the Hsp70-Bim PPI interface. These results could benefit the design of further inhibitors to occupy the Bim binding site on the Hsp70 surface. The binding mechanism of S1g-6 and Hsp70 was predicted through the molecular dynamics (MD) method by Gromacs-2021.3. The MD simulation was performed with 100-ps NVT and 100-ps NPT ensemble, and the force field was chosen as the Charmm36 force field. The temperature was set as 300K, the time step was 2fs and the total MD simulation time was 500ns.

Role of miRNAs to control the progression of Chronic Myeloid Leukemia by their expression levels.

Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder distinguished by a specific genetic anomaly known as a reciprocal translocation between chromosomes 9 and 22. This translocation causes fusion between the BCR and ABL regions. Consequently, BCR::ABL oncoprotein is formed, which plays a significant role in driving CML progression. Imatinib, a tyrosine kinase inhibitor (TKI), became the first line of drugs against CML. However, with continuous treatment, patients developed resistance against it. Indeed, to address this challenge, microRNA-based therapy emerges as a promising approach. miRNAs are 20-25 nucleotides long and hold great significance in various cellular processes, including cell differentiation, proliferation, migration, and apoptosis. In several malignancies, it has been reported that miRNAs might help to promote or prevent tumourigenesis and abnormal expression because they could act as both oncogenes/tumor suppressors. Recently, because of their vital regulatory function in maintaining cell homeostasis, miRNAs might be used to control CML progression and in developing new therapies for TKI-resistant patients. They might also act as potential prognostic, diagnostic, and therapeutic biomarkers based on their expression profiles. Various annotation tools and microarray-based expression profiles can be used to predict dysregulated miRNAs and their target genes. The main purpose of this review is to provide brief insights into the role of dysregulated miRNAs in CML pathogenesis and to emphasize their clinical relevance, such as their significant potential as therapeutics against CML. Utilizing these miRNAs as a therapeutic approach by inhibition or amplification of their activity could unlock new doors for the therapy of CML.

Cell-penetrating peptide and cationic liposomes mediated siRNA delivery to arrest growth of chronic myeloid leukemia cells in vitro

Gene silencing through RNA interference (RNAi) is a promising therapeutic approach for a wide range of disorders, including cancer. Non-viral gene therapy, using specific siRNAs against BCR-ABL1, can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKIs) therapies, given the prevalence of clinical TKI resistance. The main challenge for such approaches remains the development of the effective delivery system for siRNA tailored to the specific disease model.The purpose of this study was to examine and compare the efficiency of endosomolytic cell penetrating peptide (CPP) EB1 and PEG2000-decorated cationic liposomes composed of polycationic lipid 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2Х3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for anti-bcr-abl siRNA delivery into the K562 human CML cell line. We show that both EB1 and 2Х3-DOPE-DSPE-PEG2000 (0.62 % mol.) liposomes effectively deliver siRNA into K562 cells by endocytic mechanisms, and the use of liposomes leads to more effective inhibition of expression of the targeted gene (BCR-ABL1) and cancer cell proliferation. Taken together, these findings suggest that PEG-decorated cationic liposomes mediated siRNA delivery allows an effective antisense suppression of certain oncogenes, and represents a promising new class of therapies for CML.

Long-term results of therapy for chronic myeloid leukemia: a 20-year analysis of the use of tyrosine kinase inhibitors in Russia

Introduction. In Russia, within the framework of the GIPAP program, in the period from 2001 to 2007 at the National Medical Research Center for Hematology, imatinib therapy was initiated in 235 patients in the chronic phase of chronic myelogenous leukemia (CML).Aim: to analyze the long-term results of therapy in patients with CML who started imatinib therapy as part of the GIPAP program.Methods. A retrospective analysis of the results of therapy was performed in 235 patients with СР CML, who received imatinib under the GIPAP program from 2001 to 2007 at the National Medical Research Center for Hematology. The protocols for therapy and monitoring of the residual disease of patients at various time intervals were determined by the clinical recommendations relevant at that time in the conditions of real clinical practice and the possibilities of the patient’s region of residence. Overall survival and survival without discontinuation of imatinib therapy, univariate and multivariate analysis of overall survival were performed. The cumulative incidence of responses was calculated. An analysis of response factors, the probability of death from concomitant diseases and death from CML was carried out.Results. The median follow-up of living patients at the time of analysis was 17.3 years (IQR 15.5–18.5). 70 (30 %) patients died, with the median time to death from the start of therapy being 7.8 years (IQR 3.7–13.6). The overall 10-year, 15-year and 20-year survival rates were 82 %, 74 % and 62 %. The cause of death in 43 cases (61%) was the progression of CML to the phase of acceleration or blast crisis and death out of remission for an unspecifi ed cause. 27 (39%) patients died from causes not related to CML. Patient age at initiation of imatinib therapy, length of time from diagnosis to initiation of imatinib therapy, and Sokal and ELTS risk groups at disease onset were identifi ed as signifi cant for survival by univariate analysis. Multivariate analysis showed independent predictive value for overall survival for age at initiation of imatinib therapy, length of illness before imatinib treatment, and ELTS risk group at disease onset. Among patients who died from CML progression, the proportion of patients who did not achieve CCyR for the entire period of therapy before death was 83% (35/42), while among patients who died from concomitant diseases, the proportion of patients without CCyR for the entire period of therapy was 11 % (p < 0.0001). The median duration of imatinib therapy was 11.4 years (0.8–21 years). 40 people died during imatinib therapy, 103 patients are alive and continue therapy with imatinib, 92 patients received at least one second-generation of Tyrosine kinase inhibitors (TKI) (TKI2), of which 62 people are alive and continue treatment with TKI. No more than two lines of TKI therapy were received by 49 (21 %) patients, and three or more lines were prescribed to 43 (18 %) patients. The median duration of therapy after switching to TKI2 was 7.8 years (0.1–15.6 years). Overall 15-year survival after switching to TKI2 was 59 %. On therapy with imatinib, during the entire observation period, complete cytogenetic response (CCyR) was achieved in 171 patients (73 %), another 18 patients (8 %) achieved CCyR for the fi rst time after switching to TKI2. Major (MMR) and deep molecular response (DMR) were achieved with imatinib in 129 (56 %) and 124 (53 %) patients, with TKI2 TKI2 therapy in 38 (16 %) and 33 (14 %) patients, respectively. Multivariate analysis showed an independent predictive value of only the time period from diagnosis to the start of imatinib treatment for achieving molecular responses to TKI therapy.Conclusion. After 20 years of monitoring patients on TKI therapy, we still cannot say that survival in CML is comparable to the survival of normal population. Long-term follow-up confi rms the fact that tumor reduction to at least the level of CCyR is the most signifi cant surrogate marker associated with a reduced risk of death from CML. Timely diagnosis of the disease, rapid initiation of targeted therapy and the fastest possible induction of cytogenetic and molecular responses is a very important mechanism for reducing the risk of resistant course and progression of CML.

Assessment of knowledge, attitudes, and practices of CML patients and their families toward TKI therapy in China.

The introduction of tyrosine kinase inhibitors (TKIs) has revolutionized chronic myeloid leukemia (CML) treatment. The knowledge, attitude, and practice (KAP) of patients and their families play a significant role in treatment adherence and effectiveness. This study aimed to investigate the KAP of CML patients and their families regarding TKI therapy in China. From November 1 to December 31, 2022, a cross-sectional study was conducted at the Affiliated Huai'an No. 1 People's Hospital in China. A total of 313 CML patients and 268 family caregivers were selected using convenience sampling. Participants answered a self-designed questionnaire. The questionnaire contained demographic/clinical data and assessed KAP toward CML and TKI therapy. Participants exhibited mean KAP scores of 8.91 (55.7%), 33.10 (73.6%), and 2.20 (73.3%), respectively. Family members had higher knowledge and practice scores than patients (both P < .05), with factors such as younger age, urban residency, higher education, employment, higher income, and interaction with peers correlating with better knowledge scores (P < .001). Although participants were well-informed about their diagnosis and medication (>80%), understanding of disease causes (<30%) and treatment prognosis and side effects (<50%) was limited, and cost concerns affected 80.55%. Anxiety and depression were reported more among caregivers (46.64% and 13.8%) than patients (29.71% and 11.51%). While 84.85% adhered to the doctor's instructions, only 68.50% actively sought more CML information. Positive correlations were observed among KAP scores, indicating their interdependence (knowledge-attitude: R = 0.397; knowledge-practice: R = 0.598; attitude-practice: R = 0.353; all P < .001). The findings underscore the importance of tailored education to fill knowledge gaps about CML and the need to address financial concerns and provide psychological support. The positive correlations among knowledge, attitudes, and practices emphasize the need for comprehensive interventions. In conclusion, this study highlights the importance of tailored education, addresses financial concerns, and provides emotional support for CML patients and caregivers in China, despite limitations such as convenience sampling and questionnaire design. Future research should evaluate the effectiveness of educational interventions and long-term outcomes to further enhance the overall well-being of this population.

HJKC3-007: Outcomes of Patients with Chronic Myeloid Leukemia in Chronic Phase Treated with Asciminib and Other Tyrosine Kinase Inhibitors in Routine Clinical Practice

Background and significance: There are typically high response rates to first line TKI therapies in chronic phase chronic myeloid leukemia (CP-CML). However, responses in later lines (second line and beyond) are diminished and highly dependent on the specific tyrosine kinase inhibitors (TKIs) used. Asciminib received FDA approval in October 2021 for the treatment of adult patients with Philadelphia chromosome-positive (Ph+) CML-CP who were previously treated with two or more TKIs, and in patients with Ph+ CML-CP with the T315I mutation. Our goal in this study is to evaluate the role of asciminib therapy for patients with CML beyond clinical studies, in routine clinical practice. Study Design and methods: Data for this study will be abstracted from the HJKC3 (H. Jean Khoury Cure CML Consortium) CML registry project (HJKC3-0005); the HJKC3 CML Registry is a collaborative academic multi-site registry. Patients will be consented to allow collection of both retrospective and prospective data for the HJKC3 CML Registry. The data forms and surveys will be built and managed through the Research Electronic Data Capture (REDCap), a secure web application managed by the Clinical &amp; Translational Science Institute. Participating sites include members of the HJKC3 (curecml.org). This study will be an opportunity to evaluate asciminib usage in ‘real-world’ academic practices across the United States. This observational study will include cohorts of patients diagnosed with CP-CML previously treated with two or more tyrosine kinase inhibitors and/or receiving asciminib and/or those with a T315I mutation, not currently enrolled in a randomized controlled trial. The HJKC3 CML Registry intends to collect clinical and patient reported outcome (PRO) data. After obtaining patient consent, patients will be invited to complete PRO assessments. The study will attempt to collect PRO data regardless of disease or treatment status. PRO assessments will be primarily administered online, though study staff will offer alternatives (e.g., phone) for participants without internet access. Study subjects will be emailed a link to access a secure platform hosted by one of the study sites, Medical College of Wisconsin (MCW), using the REDCap platform. Study Objectives: The primary objective of the study is to describe characteristics of patients who were previously treated with two or more TKIs and treated with asciminib or with any other TKIs. The secondary objectives are to describe the clinical outcomes and PRO of those patients. Exploratory objectives include description of characteristics, treatment outcome and PRO for patients with T315I mutation. Statistical plan: The sample size of approximately 225 participants during the 5-year recruitment period is based on the available clinical expertise and ongoing recruitment data in the HJKC3 CML registry in lieu of statistical power analysis. We estimate that 150 patients in the HJKC3 CML registry will be treated with other TKI and 75 treated with asciminib. Among a total of 225 patients, we estimate that 75 patients with the T315I mutation will be enrolled in the registry. No formal statistical inference will be drawn to compare the outcomes between these two groups. Bayesian predictive probability (PP) will be utilized for the exploratory analysis of Major Molecular Response (MMR). Survival probabilities will be computed using the Kaplan-Meier method, and the p-values from the log-rank test will be provided. Different PRO domains of the patient's symptoms and functioning will be analyzed separately (e.g., depression separate from fatigue, separate from GI symptoms, etc.) since combining them into a summary score may dilute the effects of the individual (and not necessarily related) components and mask actual change. The main objective for the analyses of PRO data from the registry is to describe any differences by treatment type in patients treated with asciminib and other TKIs.

Restoration of Mir-185 in Combination with BCR-ABL Downregulation By Therapeutic Delivery of siRNA with Lipid Nanoparticle Carriers Targets Drug Resistant Leukemic Stem/Progenitor Cells

Background: Despite development of effective tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia (CML), TKI therapy is not curative as some patients develop drug resistance or are at risk of disease relapse. Importantly, CML leukemic stem cells (LSCs) remain largely unresponsive and represent a key population for sustaining disease and relapse. We previously demonstrated that miR-185 levels were significantly reduced in CD34 + stem/progenitor cells from TKI-nonresponders compared to TKI responders. Further, restoration of miR-185 levels in CML LSCs and their progenitors impaired their survival and sensitized them to TKI in vitro and in vivo. However, the therapeutic potential of downregulating BCR-ABL using siRNA in combination with miR-185 mimic treatment to target CML LSCs have yet to be elucidated. Objective: Downregulation of BCR-ABL using siRNA represents a promising therapeutic approach to inhibit its kinase and non-kinase functions to combat drug-resistant CML and overcome limitations of current therapy. Additionally, previous work suggests that restoring miR-185 levels may improve therapeutic outcomes from complimentary therapies in CML, particularly in combination with BCR-ABL inhibition. Therefore, we aim to investigate the therapeutic outcomes and mechanism of action of inhibiting BCR-ABL using siRNA in combination with miR-185 mimic treatment in drug-resistant CML. Methods: To assess the effects of BCR-ABL downregulation in combination with miR-185 restoration, we encapsulated siRNA targeting BCR-ABL and a miR-185 mimic using an optimized lipid nanoparticle (LNP) formulation. Biological effects and mechanistic changes of this novel RNA combination was evaluated using various CML cell lines expressing b2a2, b3a2, T315I mutated BCR-ABL and CD34 + stem/progenitor cells from non-responder patients. Results: A total of 7 siRNA sequences targeting various fusion regions of BCR-ABL were designed and tested to effectively target primitive CML cells. The highest performing BCR-ABL siRNA encapsulated showed significant knockdown in various CML models expressing b2a2, b3a2 and T315I mutated BCR-ABL (65-99% knockdown). After 48 hours of 0.5 μg/mL BCR-ABL siRNA LNP treatment, BCR-ABL protein levels and its direct downstream target p-CrkL are undetectable by immunoblotting. The robust downregulation of BCR-ABL results in a dose-dependent decrease in cell viability (down to 28% and 9.4% viable cells at a dose of 1 μg/mL) and increase in apoptotic cells (70% and 78% Annexin-V positive) in imatinib resistant K562 and T315I mutated cells, respectively after 72 hours. Similarly, encapsulated miR-185 mimic downregulates its main downstream target PAK6 transcript levels down to 50% in drug sensitive and drug-resistant cell lines and in TKI nonresponder CD34 + stem/progenitor cells. More importantly, our RNA LNP formulation demonstrated significant uptake (&amp;gt; 80% Cy5 positive) of fluorescent-labelled siRNA in TKI nonresponder CD34 + stem/progenitor cells and decreased BCR-ABL mRNA transcript levels by 60%. The combined BCR-ABL inhibition and miR-185 mimic treatment were determined to be highly synergistic when analysed using the Chou-Talalay (combination index less than 1), ZIP, Bliss and HSA methods (synergy scores larger than 10) in both drug-sensitive and drug-resistant CML cells. Furthermore, treatment of BCR-ABL siRNA and miR-185 using LNPs decreased the colony forming units of nonresponder patient cells by 50%. Interestingly, this effect was further enhanced when combined with TKIs, decreasing colony forming units to 20% compared to control after 14 days. Conclusion: We have uncovered a LNP formulation that is able to effectively deliver therapeutic BCR-ABL siRNA and miR-185 mimic into primitive CML cells. This combination treatment significantly reduces the levels of oncogenic BCR-ABL and PAK6, decreasing the viability clonogenic potential and increases apoptosis in CML cell line models and primary patient cells. Importantly, this combination strategy overcomes TKI resistance in CD34 + stem/progenitor cells suggesting a feasible and effective model for patients suffering from clinical resistance and relapse.

Chimeric Antigen-Receptor (CAR)-Engineered Natural Killer (NK) Cells Targeting Chronic Myeloid Leukemia (CML) Blast Crisis

Background The use of tyrosine kinase inhibitors (TKIs) has dramatically modified the therapy of chronic myeloid leukemia (CML), generating durable remissions and prolonging survival in TKI-responders. However, progression to blast crisis (BC) still occurs especially in TKI-resistant patients and represents a clinical challenge. We and others have identified IL2RA/CD25 as a typical cell surface marker of BC-CML and reported that its overexpression is correlated with the progression of CML from CP-CML to BC-CML (Imeri et al, Cells 2023). Here we show the experimental development of a third-generation CAR-NK therapy strategy against the CD25 based on the scFV of the clinically approved monoclonal humanized antibody, Basiliximab. Methods: As NK cell model, we have used the NK92 cell line which has a well-established and clinically demonstrated NK cell activity. We have lentivirally transduced NK92 cells with the CAR construct containing a selectable gene (GFP). After FACS-sorting of GFP-positive cells, phenotypical characterization was performed by FACS. The expression of the CAR-CD25 at the surface of the cells was demonstrated an anti-Fab antibody and double-positive (Fab/GFP) cells were further purified. The functionality of the cells was evaluated using CD107a degranulation assay after 3h co-culture and ELISA for IFN-gamma release. We have in parallel engineered K562 cells expressing CD25 by lentiviral transduction (K562-CD25) as well as also a second target cell line (RAJI) using the same strategy. Annexin V staining of target K562 cells was used for in vitro cytotoxicity assessments. For in vivo assays, NSG mice were intraperitoneally (IP) injected with K562-CD25 cells expressing Luciferase at Day-3 (3.10 6 cells/mouse, n = 13). At Days 0, 3, and 7, mice were treated by IP injection of either irradiated CD25 CAR-NK92 cells (10 .10 6/mouse n=6) or irradiated Wild-type (WT)-NK92 cells (n=5). The clinical evolution of mice transplanted mice was followed weekly by luminescence (IVIS 200). Results: After cell sorting, we obtained more than 90% of double-positive NK92 CAR+/GFP+ cells. Lentiviral transduction did not affect the activatory or inhibitory signals of NK92 cells. No statistical differences were observed between CD25 CAR-NK92 and WT- NK92 cells for the expression of NKp30, NKp46, KirDl2-3, TIGIT, and DNAM. However, we observed a strong increase in the Granzyme B and Perforin in CD25 CAR-NK92 cells after co-culture with K562-CD25 as compared to WT NK92 cells (p&amp;lt;0.001). Importantly, we have found increased levels of degranulation after co-culture of target K562-CD25 with CD25-CAR-NK92 cells (40%) as compared to cells co-cultured with WT NK92 alone (20% ) suggesting strongly the occurrence of an additional specific effect due to CAR-CD25. IFN-gamma levels after co-culture of CAR CD25 NK92 cells were also found to be significantly increased ( 400 pg/ml) in as compared to co-cultures of target cells with WT-NK92 (200 pg/ml)(p&amp;lt;0.0001). Similarly,in vitro cytotoxicity assays showed induction of higher levels of apoptosis in target cells (K562-CD25 and Raji-CD25) when co-cultured with CD25 CAR-NK92 as compared to NK92 WT (p&amp;lt;0.0001). In in vivo experiments, we have analyzed K562-CD25 leukemia-bearing mice treated with CAR-NK92 cells (n=6) or WT-NK92 cells (n=5). These experiments analyzed at D+30 post-transplant showed stronger anti-leukemia effect of CAR-NK therapy by IVIS imaging with a survival rate of 84% for mice treated with CD25 CAR-NK92 versus 40% for those treated with WT-NK92. All control mice transplanted with K562-CD25 cells and left untreated died by D+20. Conclusion: We show here for the first time the potential use of an NK cell-mediated CAR therapy strategy targeting CD25 which has been shown to be upregulated in CML blast crisis. The experimental data show a significantly increased and selective in vitro and in vivo cytotoxicity of CD25 CAR-NK92 cells against CD25-expressing leukemia cells as compared to WT-NK92 cells. These results suggest that targeting CD25 by a CD25 CAR based on Basilixiamb's scFV might be an interesting tool in BC-CML and in all acute leukemias overexpressing CD25. In order to translate these findings to NK cells derived from induced pluripotent stem cells (iPSCs), we have produced iPSCs expressing CAR-CD25 constructs and experiments are underway to evaluate the therapeutic potential of iPSC-derived CD25 CAR-NK cells in CML blast crisis or AML models.