Response In Chronic Myeloid Leukemia Research Articles

Peripheral blood quantitation of CD26 positive leukemic stem cells as a predictor of tyrosine kinase inhibitor response in chronic myeloid leukemia.

Leukemic stem cells (LSCs) are the transcriptionally low/silent cells which are resistant to the tyrosine kinase inhibitor. These have been found to play a pivotal role in disease relapse in chronic myeloid leukemia (CML) cases. The present study evaluated the correlation of absolute CML-LSC count in the peripheral blood (PB) at diagnosis and achievement of major molecular response (MMR) at 12 months in patients of CML-CP. This was a prospective, observational, non-interventional single center study including newly diagnosed adult (>18 yrs) CML-CP patients. Absolute CD26 + CML-LSC quantification was done by multiparametric flow cytometry. Patients were treated with Imatinib treatment and subsequently monitored at 3-month intervals for BCR::ABL transcript levels. MMR was defined as a BCR::ABL1 transcript level of less than 0.1% on international scale. A total of 89 patients were enrolled in the study out of which 40.5% achieved MMR at 12 months. There was a significant difference in the median absolute CML-LSC count of the patients who achieved MMR at 12 months as compared to those who did not (58.5 vs 368.1 cells/μL; p value <0.001). Using a ROC analysis, a count of <165.69 CML LSC/μL was identified to have a sensitivity of 83.8% and specificity of 72.4%, in predicting the MMR at 12 months. Absolute CML-LSC count at diagnosis in the PB predicts the MMR achievement at 12 months. An absolute count of less than 165 cells/μL is highly predictive of achieving MMR at 12 months.

Impact of CYP3A4 rs2242480 polymorphisms on Imatinib HPLC determined trough concentration and response in Chronic Myeloid Leukaemia in Pakistani patients

Objective To ascertain the impact of CYP3A4 polymorphism on the therapeutic outcome of imatinib and its trough concentration Study design Descriptive analytical study Place and duration of the study This study was conducted in the Department of Pharmacology &amp; Therapeutics, Islamic International Medical College, between December 2020 and February 2022, in collaboration with the Institute of Biomedical Genetic Engineering, KRL Hospital Islamabad. Methods Patients with Chronic Myeloid Leukaemia age range–18-70 years were included in this study. One group comprised responders and the other group comprised non-responders. Imatinib trough levels in both groups were determined using High-performance Liquid Chromatography and compared, and the association was determined with therapeutic outcomes. DNA was extracted, and the PCR restriction fragment length polymorphism technique was used to identify the alleles. The results were analyzed using Statistical Package for Social Sciences (SPSS) version 22.0. Results The imatinib concentration in patients with the homozygous wild allele CC of rs2242480 was higher(1298ng/ml) as compared with the mutant homozygous TT allele (489ng/ml) or heterozygous allele CT(873ng/ml) Conclusion There was a significant association between imatinib trough levels and CYP3A4 polymorphism, and trough concentration was found to be lower in patients with the TT or CT variant of rs2242480 than in patients with the CC genotype.

Abstract 2307: State-transition modeling of blood transcriptome predicts disease evolution and treatment response in chronic myeloid leukemia (CML)

Abstract Chronic myeloid leukemia (CML) is initiated and initially maintained by BCR-ABL which is clinically targeted using tyrosine kinase inhibitors (TKIs). Although TKIs can induce long-term remission, they are frequently not curative. Thus, CML is an ideal system to test our hypothesis that transcriptome-based state-transition models accurately predict cancer evolution and treatment response. We collected time-sequential blood samples from tetracycline-off (Tet-Off) BCR-ABL-inducible transgenic mice that recapitulates human chronic phase CML and wild-type controls. From the transcriptome, we constructed a CML state-space and a three-well leukemogenic potential landscape that describes CML state-transition from health to disease. The potential’s stable critical points were used to define three distinct disease states. The Early state was characterized by anti-CML genes (n=35) that opposed leukemia progression, whereas an expanding number of pro-CML genes characterized the Transition state (n=357) and Late states (n=1,858). Gene modules that were co-regulated at the unstable points in the potential landscape were identified as the drivers of transition between stable disease states. To investigate CML therapies we used two treatments: silencing of BCR/ABL by readministering Tet (Tet-on, Tet-off [TOTO]) which represented a best-case scenario of disease being cured, and TKI (nilotinib) therapy which represents current clinical practice. TOTO returned the diseased mice transcriptomes to a near health state, without reaching it, suggesting partly irreversible transformation. TKI, however, only reverted the transcriptome to an intermediate disease state, without approaching health, and disease relapse occurred soon after treatment. Finally, using only the earliest time-point as initial conditions, our state-transition models accurately predicted both disease progression and treatment response to both therapies for each individual mouse. In conclusion, these results show state-transition analysis is a valuable approach to gain real time insights into CML development, progression and ongoing treatment response that can also be applied to other type of leukemia and cancers in general. We predicted disease evolution and treatment response in a murine model that recapitulates human disease, supporting this as a potentially valuable approach to time clinical intervention even before phenotypic changes become detectable. Citation Format: David E. Frankhouser, Russell C. Rockne, Dandan Zhao, Sergio Branciamore, Lisa Uechi, Denis O'Meally, Yu-Hsuan Fu, Ya-Huei Kuo, Bin Zhang, Guido Marcucci. State-transition modeling of blood transcriptome predicts disease evolution and treatment response in chronic myeloid leukemia (CML) [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 2307.

State-transition modeling of blood transcriptome predicts disease evolution and treatment response in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is initiated and maintained by BCR::ABL which is clinically targeted using tyrosine kinase inhibitors (TKIs). TKIs can induce long-term remission but are also not curative. Thus, CML is an ideal system to test our hypothesis that transcriptome-based state-transition models accurately predict cancer evolution and treatment response. We collected time-sequential blood samples from tetracycline-off (Tet-Off) BCR::ABL-inducible transgenic mice and wild-type controls. From the transcriptome, we constructed a CML state-space and a three-well leukemogenic potential landscape. The potential’s stable critical points defined observable disease states. Early states were characterized by anti-CML genes opposing leukemia; late states were characterized by pro-CML genes. Genes with expression patterns shaped similarly to the potential landscape were identified as drivers of disease transition. Re-introduction of tetracycline to silence the BCR::ABL gene returned diseased mice transcriptomes to a near healthy state, without reaching it, suggesting parts of the transition are irreversible. TKI only reverted the transcriptome to an intermediate disease state, without approaching a state of health; disease relapse occurred soon after treatment. Using only the earliest time-point as initial conditions, our state-transition models accurately predicted both disease progression and treatment response, supporting this as a potentially valuable approach to time clinical intervention, before phenotypic changes become detectable.

CFL1 is Implicated in Chronic Myeloid Leukemia Response during Imatinib Therapy.

Cofilin (CFL1) is one critical member of the actin deploy family (ADF). Overexpression of CFL1 is associated with aggressive features and poor prognosis in malignancies. We evaluated the expression of CFL1 in patients with chronic myeloid leukemia in the chronic phase (CML-CP), acute myelocytic leukemia (AML) and healthy controls. The role of CFL1 in imatinib therapy was also investigated using cell line. We found that the expression of CFL1 was lower in CML patients than that in healthy controls, and was significantly upregulated after imatinib therapy (p<0.05). CML patients with lower CFL1 achieved higher Major molecular response (MMR) rate after 6 months of imatinib therapy (p<0.05). Cofilin, P-cofilin and F-actin, especially branched F-actin were all upregulated after imatinib therapy. The lower CFL1 expression before treatment may predicts a better response to imatinib. Imatinib affects F-actin remodeling in CML patients by regulating CFL1 expression and activity.

Single-Cell Analysis of Immune Recognition in Chronic Myeloid Leukemia Patients Following Tyrosine Kinase Inhibitor Discontinuation

Background: Modern treatment goal for patients with chronic myeloid leukemia (CML) is to maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. The control of residual leukemia cells may occur with immunological mechanisms as several studies have shown that a higher proportion of CD56 dim NK cells before TKI discontinuation is associated with a better probability of TFR. In addition, CD8+ T cell abundances and phenotypes have been associated with responses to TKIs. However, the more detailed phenotypes and mechanisms and how to translate these findings to increased TFR rates have remained unknown. Methods: We analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced (scRNA+TCRαβ-seq) samples from patients with CML ( n=13, N=25), other cancers (AML n=11, CLL n=13, solid tumors n=4), and healthy ( n=7). Additionally, we profiled 90 CML and 786 healthy TCRβ-seq samples, and sorted T cells specific to leukemia-associated antigen PR1 and matched these back to the scRNA+TCRαβ-seq and TCRβ-seq data. To validate and refine our findings, we performed co-culture of primary and expanded NK cells with CML cell lines (K562 and LAMA84) with a multiplexed scRNA-seq readout. Results: In the pan-cancer scRNA+TCRαβ-seq comparison of &amp;gt;250,000 cells, CML was separated from other cancers and healthy by the high number and active phenotype NK cells. The other phenotype more pronounced in CML was CD8+ T EM/T EMRA phenotype. Most NK cells in CML had an active CD56 dim phenotype with high expression of GZMA/B, PRF1, CCL3/4 and IFNG. In the off-TKI samples from patients with TFR, these NK cells upregulated NK cell activation genes such as TNFRSF4/OX40, TNFRSF9/4-1BB, and TNFRSF18/GITR. The most important predicted interactions between NK cells and CD34+ leukemic cells were inhibitory LGALS9- TIM3 and PVR- TIGIT interactions. Following TKI discontinuation, the inhibitory markers HAVCR2/TIM3 and TIGIT decreased in patients with TFR, which was not seen in patients with relapse following TFR. Accordingly, in our co-culture data of NK cells with CML cell lines, we noted activation of NK cells to a similar phenotype as noted in ex vivo off-TKI samples. In co-culture with CML target cells, NK cells gained an activation by upregulation of genes like TNFRSF4/OX40, TNFRSF9/4-1BB, TNFRSF18/GITR and the inhibitory markers HAVCR2/TIM3 and TIGIT. In co-culture with NK cells, CML target cells upregulated the IFN-γ response pathway including elevated HLA-expression and accordingly, upregulation of LGALS9 and PVR. The CD8+ T EM/EMRA cells that were expanded in CML diagnosis were suppressed following TKI treatment and decreased following TKI discontinuation. To track anti-CML T cells, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 from the TCRb-sequenced samples. As a validation of our classifier, we noticed that anti-PR1 T cells were more prevalent in CML than in healthy ( P adj&amp;lt;0.0001) or melanoma ( P adj&amp;lt;0.0001), more expanded than similar anti-viral T cells ( P adj&amp;lt;0.0001) and enriched in bone marrow samples compared to peripheral blood ( P adj&amp;lt;0.0001). From different TKIs, dasatinib expanded anti-PR1 T cells the most ( P adj&amp;lt;0.01). In the scRNA+TCRαβ-seq data, anti-PR1 T cells were enriched in the mature, cytotoxic CD8+ T EMRA phenotype ( P adj&amp;lt;0.0001). In a patient maintaining TFR, anti-PR1 T cells had a highly cytotoxic profile ( GZMB, PRF1) throughout the therapy with upregulated NK receptors like FCGR3A/CD16 and KIR2DL2, even more so than T cells targeting other targets e.g., different viruses. In comparison, the anti-PR1 T cells in a patient with early relapse had an exhausted profile with upregulation of memory marker GZMK and exhaustion markers like LAG3 and HAVCR2/TIM3. Conclusions: With our in-depth analysis of cellular and molecular immune responses in CML, we identified the active NK cells and anti-PR1 T cells that could help maintain TFR in patients discontinuing TKI treatment. Different immune-checkpoint blockade therapies such as anti-TIM3 could be considered as future strategies to improve the TFR rates.

Calr+ Myelofibrosis/BCR-ABL Chronic Myeloid Leukemia Overlap Syndrome Treated with Asciminib

Introduction Myeloproliferative neoplasms are considered mutually exclusive, and reports of co-occurring mutations are rare. Over the past 10 years, approximately two dozen case reports have been published of co-occurring BCR-ABL positive and CALR positive patients. Here we present the case of a patient who was reported to have myelofibrosis (MF)/myeloproliferative neoplasm (MPN), later developed chronic myeloid leukemia (CML), and ultimately progressed to acute myeloid leukemia (AML). This is unique in the literature reviewed, and may be the first reported case of this overlap syndrome being treated with asciminib. Case presentation A 40-year old male was diagnosed in 2018 with MF/MPN based upon a hypercellular bone marrow biopsy with diffuse reticulin fibrosis. He was negative for JAK2, but was not tested for CALR or MPL mutations. Treatment was ruxolitinib and hydroxyurea. Two years later, the patient was diagnosed with CML after being admitted for recurrent fevers, headache, and shortness of breath. At that time, a type 2 CALR mutation (CALR K385Nfs*47) was noted. He started dasatinib and decitabine, which resulted in a drop from 28.31% BCR-ABL to 0.0196% (Table 1A). However, hyperuricemia and renal failure led to a change in regimen and the patient was started on asciminib and ropeginterferon alfa-2b-njft in January 2022. By December, the patient's BCR-ABL PCR dropped below quantification, but he received a diagnosis of AML based on a bone marrow biopsy which showed 43% blasts. He received cladribine and cytarabine, but was found to have leukemic infiltration of the spinal cord five months later. The patient was referred to hospice and passed away in June 2023. Discussion To our knowledge, this is the first MPN-CML patient reported to have been treated with asciminib. This case illustrates its effectiveness in treating CML, but highlights the need for improved treatments for CALR+ MPN. Moreover, this case illustrates the importance of testing for CALR, MPL, and BCR-ABL in addition to JAK2 immediately when a patient presents with an MPN. It is unknown whether this patient's CALR mutation preceded the BCR-ABL translocation, and there is debate in the literature with respect to mutation chronology. The patient's BCR-ABL burden dropped from 28.31% to 0.0196% while the CALR burden remained around 94% over the same period (Table 1B). Thus, it is unlikely that the BCR-ABL mutation preceded the CALR mutation within the same clone. The patient's &amp;gt;90% CALR allele burden is higher than reported in the other reported cases, where the burden ranged from 10% to 68%. Furthermore, none of the other cases were reported to progress to AML, though follow up data is lacking. The heavy allelic burden in our patient may have been a contributing factor to the development of AML, as has been reported elsewhere. The patient's overall mutational profile is of interest. Type 2 CALR mutations have been reported to have less favorable prognoses than type 1 mutations. In addition to the CALR and BCR-ABL mutations, the patient had ASXL1 A861Hfs*6, EZH2 D730*, and NRAS G12V. All three mutations are associated with unfavorable outcomes in MDS, AML, and/or CML. Genomic instability has been discussed in relation to both CML and JAK2-positive MPN. Our patient's heavy mutational burden suggests CALR-positive neoplasms may also present against a background of inherent genomic instability. Conclusion We reported a case of MPN, followed by CML, and then AML. The patient was positive for CALR and BCR-ABL. He achieved a complete response in CML after targeted treatment, with asciminib consolidating the gains the patient made on earlier therapies. However, he always maintained a high CALR burden. This patient's mutational profile and disease course argue for the importance of early testing for CALR and BCR-ABL in addition to JAK2 and MPL in the setting of an MPN and for further research into the underlying causes of these overlap conditions.

Identification of Multivariable microRNA and Clinical Biomarkers Panels to Predict Imatinib Response in Chronic Myeloid Leukemia at Diagnosis

Imatinib Mesylate (IM) was once hailed as the magic bullet for chronic myeloid leukemia (CML) and remains a front-line therapy for CML to this day alongside other tyrosine kinase inhibitors (TKIs). However, TKI treatments are rarely curative and while they demonstrate efficacy in managing the disease, patients are often required to receive life-long treatment or risk experiencing relapse. Patients also frequently harbor innate TKI-resistance mechanisms to front-line IM therapy and without prior screening, patients can spend precious months on non-optimal treatment before transitioning to other therapeutic strategies. Thus, there is a growing interest in studying biomarkers in patients which can predict TKI response upon diagnosis. CML has unique microRNA (miRNA) expression profiles at different stages of the disease and in response to TKI-treatment. We previously generated global transcriptome profiles on treatment-naïve CD34 + CML cells with known subsequent imatinib (IM) responses and identified several differentially expressed miRNAs in IM-nonresponders as compared to IM-responders. We also reported that IM response could be predicted in treatment-naïve CD34 + cells by an in vitro colony forming cell (CFC) assay. Additionally, we previously analyzed patient data from the ENESTxtnd clinical trial to show that miRNA expression combined with CFC output data could accurately predict Nilotinib response in patients. In this study, we used a Cox Proportional Hazard (CoxPH) analysis to identify 22 out of a pool of 35 pre-validated miRNAs which were associated with IM nonresponse in CD34 + CML cells of 80 patients at diagnosis. These patients were later classified as IM-responders or IM-nonresponders based on the latest European LeukemiaNet (ELN) guidelines. Interestingly, a Welch t-test revealed 16 of these 22 IM-response associated miRNAs to be differentially expressed between IM-responders and IM-nonresponders. Between IM-responders and IM-nonresponders we also found 7 clinical parameters that were associated with IM-response of which 3 matched parameters had significantly different values including CFC assay outputs ( p=0.0015), Sokal scores ( p=0.0070) and white blood cell counts (WBC, p=0.0028). We then trained a machine learning model employing the random-forest (RF), gradient-boosting (GBM) and naïve-bayes (NB) algorithms with different combinations of the 16 miRNAs with and without the clinical parameters of these patients to identify panels with high predictive performance based on area-under-curve (AUC) values of receiver-operating-characteristic (ROC) and precision-recall (PR) curves. Notably, the multivariable panel consisting of both miRNAs and clinical features (AUC-ROC RF=0.83, GBM=0.83, NB=0.84, AUC-PR RF=0.68, GBM=0.67, NB=0.70) performed better than either miRNA (AUC-ROC RF=0.72, GBM=0.73, NB=0.78, AUC-PR RF=0.49, GBM=0.51, NB=0.4) or clinical (AUC-ROC RF=0.82, GBM=0.84, NB=0.84, AUC-PR RF=0.64, GBM=0.67, NB=0.64) panels alone. Interestingly, 2 miRNAs in this panel, miR-185 and miR-145, were also significant classifiers for our Nilotinib predictive study suggesting that expression patterns of these miRNAs may have predictive properties for multiple TKI responses. Thus, we show that predictive accuracy of biomarkers may be supplemented by inclusion of multivariable parameters, and our findings may inform future studies on developing predictive panels for more optimized treatment plans in the clinic.

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4 +/+ and Lama4 -/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4 -/- recipients showed significantly increased platelet counts in blood compared to Lama4 +/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4 -/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1 + cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4 -/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4 -/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4 -/- recipients (33.6%) than that in Lama4 +/+ recipients (41.5%), raising a possibility that Lama4 -/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4 -/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.

The impact of circulating 25-hydroxyvitamin D and vitamin D receptor variation on leukemia-lymphoma outcome: Molecular and cytogenetic study

Vitamin D (VD) potentially has a crucial function in the development of cancerous cells. This study aims to detect the role of vitamin D concentration and its receptor polymorphisms as possible prognostic biomarkers in patients with leukemia/lymphoma and further will attempt to detect the presence of the Philadelphia chromosome abnormality in chronic myeloid leukemia (CML). Seventy-five patients, in addition to 50 healthy individuals were included. Three single nucleotide polymorphisms of the vitamin D receptor (FokI, Tru91, and ApaI) were identified via Polymerase Chain Reaction- Fragment Length Polymorphism (PCR-RFLP). Sanger sequencing and karyotyping for all patients has been undertaken. Out of 75 patients, 69 (92.0%) were vitamin D deficient. The homozygous genotype TT of FokI is the most commonly found in non-Hodgkin's lymphoma, while the heterozygous CT is observed markedly in CML, chronic lymphoid leukemia, and Hodgkin's lymphoma. The AC and CC genotypes of ApaI are more frequent in patients with CML, while the AC genotype is the most common in HL. In Tru9I, the GG genotype has a wider distribution in individuals diagnosed with leukemia. The PCR-RFLP and Sanger sequencing techniques together confirmed significant genotype respectively. The Philadelphia chromosome, t (9;22) was found in five (17%) cases with CML. There is a marked relationship between FokI, ApaI, and Tru91 polymorphisms and the chance of developing leukemia. In lymphoma, a significant connection between the polymorphisms of FokI and ApaI is frequently detected. Cytogenetic and molecular testing are essential for detection of CML and monitoring therapy response.

Single-cell analysis of immune recognition in chronic myeloid leukemia patients following tyrosine kinase inhibitor discontinuation

Immunological control of residual leukemia cells is thought to occur in patients with chronic myeloid leukemia (CML) that maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. To study this, we analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced samples (scRNA+TCRαβ-seq) from patients with CML (n = 13, N = 25), other cancers (n = 28), and healthy (n = 7). The high number and active phenotype of natural killer (NK) cells in CML separated them from healthy and other cancers. Most NK cells in CML belonged to the active CD56dim cluster with high expression of GZMA/B, PRF1, CCL3/4, and IFNG, with interactions with leukemic cells via inhibitory LGALS9–TIM3 and PVR–TIGIT interactions. Accordingly, upregulation of LGALS9 was observed in CML target cells and TIM3 in NK cells when co-cultured together. Additionally, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 and quantified anti-PR1 T cells in 90 CML and 786 healthy TCRβ-sequenced samples. Anti-PR1 T cells were more prevalent in CML, enriched in bone marrow samples, and enriched in the mature, cytotoxic CD8 + TEMRA cluster, especially in a patient maintaining TFR. Our results highlight the role of NK cells and anti-PR1 T cells in anti-leukemic immune responses in CML.

A conserved ZFX/WNT3 axis modulates the growth and imatinib response of chronic myeloid leukemia stem/progenitor cells

BackgroundZinc finger protein X-linked (ZFX) has been shown to promote the growth of tumor cells, including leukemic cells. However, the role of ZFX in the growth and drug response of chronic myeloid leukemia (CML) stem/progenitor cells remains unclear.MethodsReal-time quantitative PCR (RT–qPCR) and immunofluorescence were used to analyze the expression of ZFX and WNT3 in CML CD34+ cells compared with normal control cells. Short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) technologies were used to study the role of ZFX in growth and drug response of CML cells. Microarray data were generated to compare ZFX-silenced CML CD34+ cells with their controls. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to study the molecular mechanisms of ZFX to regulate WNT3 expression. RT–qPCR and western blotting were used to study the effect of ZFX on β-catenin signaling.ResultsWe showed that ZFX expression was significantly higher in CML CD34+ cells than in control cells. Overexpression and gene silencing experiments indicated that ZFX promoted the in vitro growth of CML cells, conferred imatinib mesylate (IM) resistance to these cells, and enhanced BCR/ABL-induced malignant transformation. Microarray data and subsequent validation revealed that WNT3 transcription was conservatively regulated by ZFX. WNT3 was highly expressed in CML CD34+ cells, and WNT3 regulated the growth and IM response of these cells similarly to ZFX. Moreover, WNT3 overexpression partially rescued ZFX silencing-induced growth inhibition and IM hypersensitivity. ZFX silencing decreased WNT3/β-catenin signaling, including c-MYC and CCND1 expression.ConclusionThe present study identified a novel ZFX/WNT3 axis that modulates the growth and IM response of CML stem/progenitor cells.

Identification of multivariable microRNA and clinical biomarker panels to predict imatinib response in chronic myeloid leukemia at diagnosis.

Imatinib Mesylate (imatinib) was once hailed as the magic bullet for chronic myeloid leukemia (CML) and remains a front-line therapy for CML to this day alongside other tyrosine kinase inhibitors (TKIs). However, TKI treatments are rarely curative and patients are often required to receive life-long treatment or otherwise risk relapse. Thus, there is a growing interest in identifying biomarkers in patients which can predict TKI response upon diagnosis. In this study, we analyze clinical data and differentially expressed miRNAs in CD34+ CML cells from 80 patients at diagnosis who were later classified as imatinib-responders or imatinib-nonresponders. A Cox Proportional Hazard (CoxPH) analysis identified 16 miRNAs that were associated with imatinib nonresponse and differentially expressed in these patients. We also trained a machine learning model with different combinations of the 16 miRNAs with and without clinical parameters and identified a panel with high predictive performance based on area-under-curve values of receiver-operating-characteristic and precision-recall curves. Interestingly, the multivariable panel consisting of both miRNAs and clinical features performed better than either miRNA or clinical panels alone. Thus, our findings may inform future studies on predictive biomarkers and serve as a tool to develop more optimized treatment plans for CML patients in the clinic.

Role of miRNA -150 between different BCR-ABL p210 transcript levels and between different levels of imatinib optimal response in CML patients

The dysregulation of miRNA expression patterns is one of the many effects developments of cancer, miRNA has been found to express abnormally in hematological neoplasia such as chronic myeloid leukemia and solid malignancies. Resistance and the degree of response following tyrosine kinase inhibitor treatment are correlated with miRNA expression. Hence, in this study we tried to study the relationship of miRNA-150 between different breakpoint cluster region–Abelson (BCR- ABL) P210 transcript levels and the role miRNA- 150 between different levels of imatinib optimal response in chronic myeloid leukemia (CML). Our study included sixty chronic myeloid leukemia (CML) patients they were divided into two groups based on response to imatinib therapy, thirty samples of the optimal molecular response of chronic myeloid leukemia (CML) patients, and thirty samples of failure molecular response chronic myeloid leukemia (CML) patients. Thirty samples of apparently healthy volunteers were included and evaluated as control. According to the P210 BCR-ABL%, the results showed a significant difference (P= &lt; 0.0001) between the responder and the failure response CML patients. Assessed the result of miRNA-150 showed a significant difference between both CML patients (P = &lt; 0.0001), assessed miRNA-150 level among different response groups, and failure response of CML patients (P = 0.0002). A cutoff value of response vs. failure response (1.784) with high sensitivity can be a diagnostic value to differentiate between response and failure response. Changing gene expression with different amounts of miRNAs had an impact on drug-gene interactions, with consequences for cell growth and death. Gene expression of different levels miRNA-150 among of CML patients of imatinib therapy showed high expression in response patients than failure response patients. The gene expression level of miRNA-150 differs through different responses in CML patients.

FUBP3 regulates chronic myeloid leukaemia progression through PRC2 complex regulated PAK1-ERK signalling.

The development of resistance and heterogeneity in differential response towards tyrosine kinase inhibitors (TKI) in chronic myeloid leukaemia (CML) treatment has led to the exploration of factors independent of the Philadelphia chromosome. Among these are the association of deletions of genes on derivative (der) 9 chromosome with adverse outcomes in CML patients. However, the functional role of genes near the breakpoint on der (9) in CML prognosis and progression remains largely unexplored. Copy number variation and mRNA expression were evaluated for five genes located near the breakpoint on der (9). Our data showed a significant association between microdeletions of the FUBP3 gene and its reduced expression with poor prognostic markers and adverse response outcomes in CML patients. Further investigation using K562 cells showed that the decrease in FUBP3 protein was associated with an increase in proliferation and survival due to activation of the MAPK-ERK pathway. We have established a novel direct interaction of FUBP3 protein and PRC2 complex in the regulation of ERK signalling via PAK1. Our findings demonstrate the role of the FUBP3 gene located on der (9) in poor response and progression in CML with the identification of additional druggable targets such as PAK1 in improving response outcomes in CML patients.

Loss of G0/G1 switch gene 2 (G0S2) promotes disease progression and drug resistance in chronic myeloid leukaemia (CML) by disrupting glycerophospholipid metabolism.

Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 have turned chronic myeloid leukaemia (CML) from a fatal disease into a manageable condition for most patients. Despite improved survival, targeting drug-resistant leukaemia stem cells (LSCs) remains a challenge for curative CML therapy. Aberrant lipid metabolism can have a large impact on membrane dynamics, cell survival and therapeutic responses in cancer. While ceramide and sphingolipid levels were previously correlated with TKI response in CML, the role of lipid metabolism in TKI resistance is not well understood. We have identified downregulation of a critical regulator of lipid metabolism, G0/G1 switch gene 2 (G0S2), in multiple scenarios of TKI resistance, including (1) BCR::ABL1 kinase-independent TKI resistance, (2) progression of CML from the chronic to the blast phase of the disease, and (3) in CML versus normal myeloid progenitors. Accordingly, CML patients with low G0S2 expression levels had a worse overall survival. G0S2 downregulation in CML was not a result of promoter hypermethylation or BCR::ABL1 kinase activity, but was rather due to transcriptional repression by MYC. Using CML cell lines, patient samples and G0s2 knockout (G0s2-/- ) mice, we demonstrate a tumour suppressor role for G0S2 in CML and TKI resistance. Our data suggest that reduced G0S2 protein expression in CML disrupts glycerophospholipid metabolism, correlating with a block of differentiation that renders CML cells resistant to therapy. Altogether, our data unravel a new role for G0S2 in regulating myeloid differentiation and TKI response in CML, and suggest that restoring G0S2 may have clinical utility.

Integrating Single-Cell Transcriptome and Network Analysis to Characterize the Therapeutic Response of Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by a unique BCR-ABL fusion gene. Tyrosine kinase inhibitors (TKIs) were developed to target the BCR-ABL oncoprotein, inhibiting its abnormal kinase activity. TKI treatments have significantly improved CML patient outcomes. However, the patients can develop drug resistance and relapse after therapy discontinues largely due to intratumor heterogeneity. It is critical to understand the differences in therapeutic responses among subpopulations of cells. Single-cell RNA sequencing measures the transcriptome of individual cells, allowing us to differentiate and analyze individual cell populations. Here, we integrated a single-cell RNA sequencing profile of CML stem cells and network analysis to decipher the mechanisms of distinct TKI responses. Compared to normal hematopoietic stem cells, a set of genes that were concordantly differentially expressed in various types of stem cells of CML patients was revealed. Further transcription regulatory network analysis found that most of these genes were directly controlled by one or more transcript factors and the genes have more regulators in the cells of the patients who responded to the treatment. The molecular markers including a known drug-resistance gene and novel gene signatures for treatment response were also identified. Moreover, we combined protein-protein interaction network construction with a cancer drug database and uncovered the drugs that target the marker genes directly or indirectly via the protein interactions. The gene signatures and their interacted proteins identified by this work can be used for treatment response prediction and lead to new strategies for drug resistance monitoring and prevention. Our single-cell-based findings offered novel insights into the mechanisms underlying the therapeutic response of CML.

A Suboptimal Response in CML Is Associated with Expression of the Immune Checkpoint VISTA

Introduction: Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML) leading to molecular remission in most patients. The next challenges are decreasing resistance and improving the odds of treatment-free remission. Understanding determinants of disease persistence and suboptimal responses is critical for improving these probabilities. The role of the immune system in the control or eradication of CML has been demonstrated in multiple settings including successful graft-vs-leukemia effect and the requirement of active T-cells and natural killer (NK) cells for sustained remission prior to therapy discontinuation. Herein, we aim to demonstrate a comparison of immune determinants of response in CML in those who achieve a deep molecular response (MR4.5) compared to suboptimal responders. Methods: We prospectively collected longitudinal blood and bone marrow samples from patients receiving treatment for CML and characterized them using cytometry by time of flight (CyTOF). 20 patients that achieved MR4.5 at any time during their treatment were compared to a cohort of 32 patients with suboptimal response to therapy according to the European LeukemiaNet (ELN) response milestones (Table 1). We custom-designed this CyTOF panel (36-plex) to include markers of leukemia/stem progenitor cells, lineage defining, intracellular signaling, immune exhaustion and activation markers. Samples were barcoded, pooled and processed simultaneously to overcome technical variations and batch effects. CD45hi cells (leukocytes) were grouped using PhenoGraph clustering, based on expression of these markers in the earliest samples available, collected at various timepoints during treatment. Results: Analysis of differential expression patterns revealed 25 distinct clusters. Patients with suboptimal response to therapy had a higher proportion of CD45hiCD3+ cells (T-cells) compared to those with an optimal response (median of 59% vs 53%, P=0.04). Notably, this included a higher proportion of exhausted CD8+ cytotoxic T-cells in patients with suboptimal response characterized by high Eomes and low Tbet expression (median of 8.0% vs 1.9%, P=0.04). In contrast, CD56+ NK-cells were more prevalent in the optimal response group (median of 5.5% vs 3.2%, P=0.04), particularly an interferon gamma releasing (CD56hiCD16loTbet+) subpopulation (median of 4.1% vs 2.5%, P=0.04). While CD68+ cells (macrophages) were detected at similar rates in both the optimal and suboptimal response groups (median of 16.5% vs 15.5%, P=0.07), the phagocytic subpopulation (CD16-CD14+CD68+HLA-DRlo) was more prevalent in those who achieved an optimal response (median of 15.1% vs 13.2%, P=0.02). Patients with suboptimal response had a higher proportion of CD68+HLA-DRloVISTAhi cells (median of 5.6% vs 1.8%, P=0.004), referring to macrophages expressing the immune regulatory ligand V-domain immunoglobulin suppressor of T cell activation (VISTA). Like PD-L1 and other B7 family ligands, VISTA attenuates T-cells response to antigens and VISTA overexpression has been associated with resistance to immune checkpoint inhibitors in solid tumors. Furthermore, cells characterized by CD66b+CXCR4+VISTAlo were increased in patients with optimal response (median of 20.6% vs 0.6%, P=0.01), indicating a higher proportion of mature granulocytes with low expression of VISTA in the optimal response group (Figure 1). Conclusion: We found that higher expression of the immune checkpoint VISTA, in addition to markers of T-cells exhaustion is associated with suboptimal response to therapy in CML. These findings highlight the importance of the immune microenvironment in response to targeted therapies such as TKIs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Key microRNAs As Predictive Biomarkers of Nilotinib Response in Chronic Myeloid Leukemia: A Sub-Analysis of the Enestxtnd Clinical Trial

Despite the effectiveness of tyrosine kinase inhibitors (TKIs) against chronic myeloid leukemia (CML), acquired drug resistance often leads to relapse. CML has unique microRNA (miRNA) expression profiles at different disease stages and in response to TKI-treatment. However, the predictive utility of miRNAs is not yet conclusive and there is a need to identify key biomarkers for prognostic tools to be used in the clinic. We have recently generated global transcriptome profiles on treatment-naïve CD34+ CML cells with known subsequent imatinib (IM) responses and identified several differentially expressed miRNAs, including miR-185 and miR-145, in IM-nonresponders as compared to IM-responders. We also reported that IM response could be predicted in treatment-naïve CD34+ cells by an in vitro colony forming cell (CFC) assay. In this study, we evaluated miRNA expression changes in CD34+ CML cells pre- and post-nilotinib (NL) therapy from 58 CML patients enrolled in the Canadian sub-analysis of the ENESTxtnd phase IIIb clinical trial and assessed a potential correlation between miRNA expression and sensitivity of CD34+ cells to TKIs in CFC assays, to predict NL response in these patients. CFC assays were performed by plating pre-treated CD34+ cells with methylcellulose-medium ± Imatinib (IM), NL and Dasatinib (DA) and showed that only NL was able to differentiate responses between NL-responders and NL-nonresponders (p=0.011) and predict NL response (p=0.00016) based on calculated cutoffs. Microfluidic qRT-PCR and a univariate Cox proportional hazard (CoxPH) analysis on miRNA expression profiles were then performed in CD34+cells obtained at diagnosis (BL), 1-month (M1) and 3-month post-NL treatment (M3) from 58 CML patients; this showed that 17 out of 47 miRNAs examined were significantly associated with NL response (p<0.05). Further Welch t-test analysis revealed that nine of these miRNAs were differentially expressed between NL-responders and NL-nonresponders. These miRNA candidates were then subject to a multivariate CoxPH analysis ± CFC assay data from each patient, which demonstrated that miR-145 (p=0.013) and miR-708 (p=0.009) together could improve predictive power at the BL state. Additionally, receiver-operating-characteristic (ROC) and precision-recall (PR) area-under-curve (AUC) values (1.2-fold) of performance plots generated by random forest (RF) and Naïve-Bayes (NB) machine learning algorithms were increased in this multivariate panel compared to individual miRNA variables. At M1 and M3, four miRNAs were found to be stably associated with NL response individually and a combination of miR-150 (M1 p<0.001, M3 p=0.01) and miR-185 (M1 p=0.009, M3 p=0.01) was significantly associated with treatment response in multivariate CoxPH analysis. Again, the multivariate analysis improved ROC and PR AUC values, especially for miR-185 (1.2 to 2-fold). Most interestingly, incorporation of NL-CFC output consistently increased AUC values up to 2-fold at both BL and M1/M3 time points that enhanced predictive performance. Together, these findings offer two predictive models for NL response in treatment-naïve or post-treatment CML patients, which could be developed into prognostic biomarkers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment-Free Response in Chronic Myeloid Leukemia Using Brazilian Imatinib Copies As First Line - Results from Two Prospective Clinical Trials

Introduction: Several trials have demonstrated the feasibility of tyrosine kinase inhibitors (TKI) treatment in chronic myeloid leukemia (CML) patients with a deep molecular response, but little data is available about the results using imatinib copies. Brazil has been using imatinib copies as a first-line treatment since 2013. Aim: To report the results of two Brazilian imatinib copies trials and to evaluate factors impacting treatment-free remission (TFR) and treatment-free survival (TFS) after imatinib discontinuation. Methods: From Dec-2016 until Oct-2018 (USP trial, NCT03239886) and from Oct-2020 until Apr-2021(Hemomed Trial- DIP), 51 eligible patients were included to participate in two trials of imatinib discontinuation. Inclusion criteria: age > 18 years, chronic phase, minimum of three years (USP trial) and four years (DIP) of imatinib copies therapy, a deep molecular response, defined as MR4.0 or MR4.5 sustained for more than 2 years, and a confirmatory test at the moment of the screening. Atypical transcripts were excluded. After discontinuation, patients on the USP trial were monitored by Q-PCR monthly for the first 6 months and bimonthly after 6 months for the first year and quarterly afterwards. Patients in DIP monitoring were monthly for the first year and every 2 months in the second year. Minimal criteria for IM re-initiation was loss of major molecular response (MMR) confirmed by two exams. Results: Median age was 53 years at the time of discontinuation. 58% male (USP) and 65% female (DIP). Imatinib´s therapy was 9.9 years (3-15.5 y). 52.9% had MR4.5 at the screening. TFS at 6 months was 78% (95 Cl 67-90), 12 months 66% (95 Cl 54-80) and at 24 months 54% (95 Cl 42-71). TFS by center at 12 months were 64.5 % (USP) and 67% (DIP). TFS according to previous molecular response after 24 months of follow-up (MR4 vs. MR4.5) (p =0,086). MMR recovery was present in 100% with a median time of 3.5 months. Four patients are still on TFR because the double testing of MMR loss was not confirmed. Conclusion: These two trials demonstrated the feasibility and safety of imatinib discontinuation, even using Brazilian copies, and the results were similar to that presented in another trial. The confirmatory exam to establish the loss of TFR was useful and safe. However, the results no suggest a benefit on monthly Q-PCR after six months of follow-up. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal