Philadelphia Chromosome Positive ALL Research Articles

Genetics and Diagnostic Approach to Lymphoblastic Leukemia/Lymphoma.

Our understanding of the genetics and biology of lymphoblastic leukemia/lymphoma (acute lymphoblastic leukemia, ALL) has advanced rapidly in the past decade with advances in sequencing and other molecular techniques. Besides recurrent chromosomal abnormalities detected by karyotyping or fluorescence in situ hybridization, these leukemias/lymphomas are characterized by a variety of mutations, gene rearrangements as well as copy number alterations. This is particularly true in the case of Philadelphia-like (Ph-like) ALL, a major subset which has the same gene expression signature as Philadelphia chromosome-positive ALL but lacks BCR-ABL1 translocation. Ph-like ALL is associated with a worse prognosis and hence its detection is critical. However, techniques to detect this entity are complex and are not widely available. This chapter discusses various subsets of ALL and describes our approach to the accurate classification and prognostication of these cases.

Clinical Outcome in Pediatric Patients with Philadelphia Chromosome Positive ALL Treated with Tyrosine Kinase Inhibitors Plus Chemotherapy-The Experience of a Polish Pediatric Leukemia and Lymphoma Study Group.

Simple SummaryPhiladelphia chromosome positive acute lymphoblastic leukemia (ALL Ph+) is rare in children, but outcomes are still poor. The aim of our study was to analyze the toxicity events and results of children with ALL Ph+ treated according to the EsPhALL2010 protocol (the European intergroup study of post induction treatment of Philadelphia chromosome positive ALL) in Poland between the years 2012 and 2019. Our treatment outcomes are still disappointing compared to those in other reports. Improvements in supportive care and emphasis placed on the determination of MRD at successive time points, which will impact decisions on therapy, may be required.The treatment of children with Philadelphia chromosome positive acute lymphoblastic leukemia (ALL Ph+) is currently unsuccessful. The use of tyrosine kinase inhibitors (TKIs) combined with chemotherapy has modernized ALL Ph+ therapy and appears to improve clinical outcome. We report herein the toxicity events and results of children with ALL Ph+ treated according to the EsPhALL2010 protocol (the European intergroup study of post-induction treatment of Philadelphia chromosome positive ALL) in 15 hemato-oncological centers in Poland between the years 2012 and 2019. The study group included 31 patients, aged 1–18 years, with newly diagnosed ALL Ph+. All patients received TKIs. Imatinib was used in 30 patients, and ponatinib was applied in one child due to T315I and M244V mutation. During therapy, imatinib was replaced with dasatinib in three children. The overall survival of children with ALL Ph+ treated according to the EsPhALL2010 protocol was 74.1% and event-free survival was 54.2% after five years. The cumulative death risk of the study group at five years was estimated at 25.9%, and its cumulative relapse risk was 30%. Our treatment outcomes are still disappointing compared to other reports. Improvements in supportive care and emphasis placed on the determination of minimal residual disease at successive time points, which will impact decisions on therapy, may be required.

Safety and Antitumor Effects of CD19-Specific Autologous Chimeric Antigen Receptor-Modified T (CAR-T) Cells Expressing the Inducible Caspase 9 Safety Switch (iC9-CAR19 T Cells) in Adult Acute Lymphoblastic Leukemia (ALL)

Safety and Antitumor Effects of CD19-Specific Autologous Chimeric Antigen Receptor-Modified T (CAR-T) Cells Expressing the Inducible Caspase 9 Safety Switch (iC9-CAR19 T Cells) in Adult Acute Lymphoblastic Leukemia (ALL)

Reversal of glucocorticoid resistance in paediatric acute lymphoblastic leukaemia is dependent on restoring BIM expression

BackgroundAcute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Glucocorticoids form a critical component of chemotherapy regimens and resistance to glucocorticoid therapy is predictive of poor outcome. We have previously shown that glucocorticoid resistance is associated with upregulation of the oncogene C-MYC and failure to induce the proapoptotic gene BIM.MethodsA high-throughput screening (HTS) campaign was carried out to identify glucocorticoid sensitisers against an ALL xenograft derived from a glucocorticoid-resistant paediatric patient. Gene expression analysis was carried out using Illumina microarrays. Efficacy, messenger RNA and protein analysis were carried out by Resazurin assay, reverse transcription-PCR and immunoblotting, respectively.ResultsA novel glucocorticoid sensitiser, 2-((4,5-dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenylacetamide (GCS-3), was identified from the HTS campaign. The sensitising effect was specific to glucocorticoids and synergy was observed in a range of dexamethasone-resistant and dexamethasone-sensitive xenografts representative of B-ALL, T-ALL and Philadelphia chromosome-positive ALL. GCS-3 in combination with dexamethasone downregulated C-MYC and significantly upregulated BIM expression in a glucocorticoid-resistant ALL xenograft. The GCS-3/dexamethasone combination significantly increased binding of the glucocorticoid receptor to a novel BIM enhancer, which is associated with glucocorticoid sensitivity.ConclusionsThis study describes the potential of the novel glucocorticoid sensitiser, GCS-3, as a biological tool to interrogate glucocorticoid action and resistance.

Different Effects of Pre-transplantation Measurable Residual Disease on Outcomes According to Transplant Modality in Patients With Philadelphia Chromosome Positive ALL.

Background: This study compared the effects of pre-transplantation measurable residual disease (pre-MRD) on outcomes in Philadelphia chromosome (Ph)-positive ALL patients who underwent human leukocyte antigen-matched sibling donor transplantation (MSDT) or who received unmanipulated haploidentical SCT (haplo-SCT).Methods: A retrospective study (n = 202) was performed. MRD was detected by RT-PCR and multiparameter flow cytometry.Results: In the total patient group, patients with positive pre-MRD had a higher 4-year cumulative incidence of relapse (CIR) than that in patients with negative pre-MRD (26.1% vs. 12.1%, P = 0.009); however, the cumulative incidence of non-relapse mortality (NRM) (7.4% vs. 15.9%, P = 0.148), probability of leukemia-free survival (LFS) (66.3% vs. 71.4%, P = 0.480), and overall survival (OS) (68.8% vs. 76.5%, P = 0.322) were comparable. In the MSDT group, patients with positive pre-MRD had increased 4-year CIR (56.4% vs. 13.8%, P < 0.001) and decreased 4-year LFS (35.9% vs. 71.0%, P = 0.024) and OS (35.9% vs. 77.6%, P = 0.011) compared with those with negative pre-MRD. In haplo-SCT settings, the 4-year CIR (14.8% vs. 10.7%, P = 0.297), NRM (7.3% vs. 16.3%, P = 0.187) and the 4-year probability of OS (77.7% vs. 72.3%, P = 0.804) and LFS (80.5% vs. 75.7%, P = 0.660) were comparable between pre-MRD positive and negative groups. In subgroup patients with positive pre-MRD, haplo-SCT had a lower 4-year CIR (14.8% vs. 56.4%, P = 0.021) and a higher 4-year LFS (77.7% vs. 35.9%, P = 0.036) and OS (80.5% vs. 35.9%, P = 0.027) than those of MSDT. Multivariate analysis showed that haplo-SCT was associated with lower CIR (HR, 0.288; P = 0.031), superior LFS (HR, 0.283; P = 0.019) and OS (HR, 0.252; P = 0.013) in cases with a positive pre-MRD subgroup.Conclusions: Our results indicate that the effects of positive pre-MRD on the outcomes of patients with Ph-positive ALL are different according to transplant modality. For Ph-positive cases with positive pre-MRD, haplo-SCT might have strong graft-vs.-leukemia (GVL) effects.

Effect of Dasatinib Vs Imatinib in the Treatment of Pediatric Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: A Randomized, Open-Label, Multicenter Study of the Chinese Children's Cancer Group

OBJECTIVE To determine whether dasatinib given at 80 mg/m2 is more effective than imatinib at 300 mg/m2 to improve event-free survival of children with Philadelphia chromosome-positive ALL, in the context of intensive chemotherapy without prophylactic cranial irradiation. DESIGN, SETTING, AND PARTICIPANTS This open-label phase III randomized study was conducted at 20 hospitals in China. Enrollment began in January 2015 and randomization was stopped in October 2018 when the early stopping criterion of the trial was met. Patients aged between 0 and 18 years were recruited. Of the 225 patients with the diagnosis, 35 declined and 1 died before treatment. INTERVENTIONS Patients were randomized to receive daily dasatinib (n=92) or imatinib (n=97) continuously for the entire duration of ALL therapy from the time of diagnosis made during remission induction to the end of continuation therapy. MAIN OUTCOMES AND MEASURES The primary outcome was event-free survival, analyzed by intent-to-treat. The secondary outcomes were relapse, toxic death, and overall survival. RESULTS With a median follow-up of 26.1 months (IQR 16.3-34.1), the 4-year event-free survival rate was 71.0% (95% CI 56.2-89.6) in the dasatinib group and 48.9% (95% CI 32.0-74.5, p=0.005) in the imatinib group (hazard ratio 2.36, 95% CI 1.27-4.40, p=0.007). The 4-year cumulative risk of any relapse was 19.8% (95% CI 4.2-35.4) in the dasatinib group and 34.4% (95% CI 15.6-53.2) in the imatinib group (p=0.01), while the 4-year cumulative risk of an isolated central-nervous-system relapse was 2.7% (95% CI 0.0-8.1) in the dasatinib group and 8.4% (95% CI -1.2-15.6) in the imatinib group (p=0.06). There were no significant differences in the frequency of severe toxicities between the two treatment groups. CONCLUSION AND RELEVANCE Intensive chemotherapy including dasatinib at 80 mg/m2 per day yielded superior results in the treatment of Philadelphia chromosome-positive ALL compared to imatinib at 300 mg/m2 per day and provided excellent control of central-nervous-system leukemia without the use of prophylactic cranial irradiation. Disclosures No relevant conflicts of interest to declare.

Targeted Treatment and Survival Following Relapse after Allogeneic Hematopoietic Cell Transplantation for Acute Leukemia and MDS in the Contemporary Era

Introduction: Relapse is the most frequent cause of treatment failure after allogeneic hematopoietic cell transplantation (alloHCT). While transplant-related mortality has decreased substantially over the last few decades, little progress has been made in outcomes and no standard of care exists for patients (pts) with post-alloHCT relapse. In the recent era, several new therapies, including targeted agents, have been approved for ALL, AML, and MDS. We conducted a study to evaluate outcomes of pts with these diseases who relapse after alloHCT in the contemporary period with routine availability of these newer therapeutic agents. Methods: We performed a single-institution retrospective cohort study to review treatment strategies and outcomes of relapse post-alloHCT. We identified 420 adult pts who received their first alloHCT in 2010-2018 using any conditioning regimen or donor source. Overall, 115 (27%) pts experienced relapse (ALL=17/64 [27%], AML=67/242 [28%], MDS=31/114 [27%]) and were included in the analysis. Results: Myeloablative (54%) matched-unrelated donor grafts (50%) were the most common types of HCTs. Peripheral blood stem cell graft (49%) and bone marrow graft (48%) were used the most. Median time from alloHCT to relapse was 5 (range 1-65) months, and 83% of relapses occurred within the first year. Only 24% and 11% of pts experienced grade II-IV acute and any chronic GVHD prior to relapse, respectively. Seven of 17 pts had Philadelphia chromosome positive ALL. Mutation panel was tested in 56% of AML and MDS. Median follow-up period after relapse was 19 (range 6-80) months. The estimated survival after relapse for all diseases was 32% (95% CI 24-41%) at 6 months, 21% (14-28%) at 12 months, and 14% (8-21%) at 24 months (Fig 1). Excluding pts treated with supportive care only, the majority received a combination of different treatments; pts with ALL received median 3 (range 1-5), pts with AML received median 2 (1-4), and pts with MDS received median 1 (1-3) agent. Targeted therapies used for ALL pts included blinatumomab (n=5) and BCR-ABL targeting tyrosine kinase inhibitors with (n=2) or without (n=4) chemotherapy. Among AML pts, targeted agents were used in 15 pts (sorafenib [n=7], 2 each with enasidenib, gemtuzumab ozagamicin, and ivosidenib, and 1 each with venetoclax and SEL24 [a dual pan-PIM/FLT3 inhibitor]). One pt each was treated with enasidenib, gemtuzumab ozagamicin, and PTC299 (an inhibitor of VEGFA mRNA translation) followed by SEL24 for MDS. Second alloHCTs (n=5) were performed median 5 (range 1-16) months after first HCT and median 1 month (range 0-5 months) after relapse. Two pts received no bridging therapy, while 3 pts received chemotherapy (n=2) or donor lymphocyte infusions (DLI [n=1]) prior to the second transplant. DLI without second transplant was used in 25 pts at a median of 20 (range 3-18) months after ALL relapse, median 2 (range 0-13) months after AML relapse, and median 3 (range 1-5) months after MDS relapse. Following DLI, 53% pts developed GVHD. Targeted therapy was associated with a trend towards better survival compared to other therapies (Fig 2, HR 0.65, 95% CI 0.41-1.03, p=0.06). Based on multivariable analysis, matched unrelated (vs. matched sibling, HR 1.70, p=0.027) or haploidentical donor grafts (HR 2.69, p=0.003), presence of grade II-IV acute GVHD before relapse (HR 2.46, p&lt;0.001), and less than 12 months from HCT to relapse (&lt;6 vs. &gt;12 months, HR 6.34, p&lt;0.001; 6-12 vs. &gt;12 months, HR 3.16, p=0.005) were adverse prognostic features with survival after relapse post-alloHCT (Table 1). Conclusion: Outcomes of pts with ALL, AML, and MDS who relapse following alloHCT remain poor in the contemporary era when several newer therapies, including targeted agents, are available for their treatment. Targeted agents were used only in a minority of post-alloHCT relapses likely due to the combination of pt status, absence of the target mutation, the agents' availability, and other factors. Pts who developed grade II-IV acute GVHD and had shorter "disease-free" duration from unrelated or haploidentical donor grafts had the significantly shorter survival following relapse. More innovative treatment strategies to prevent and treat relapse post-alloHCT are needed. Disclosures Hill: Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celegene: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; TG therapeutics: Research Funding; Genentech: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Takeda: Research Funding. Anwer:In-Cyte: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Majhail:Atara Bio: Consultancy; Anthem, Inc.: Consultancy; Nkarta: Consultancy; Mallinckrodt: Honoraria; Incyte: Consultancy.

A Fast and Accurate Diagnostic Method for Ph-like ALL Using the Ncounter System

Ph-like acute lymphoblastic leukemia (also known as BCR-ABL1-like ALL) is a new disease entity of B-cell ALL (B-ALL) that exhibits a mRNA expression profile similar to that of Philadelphia chromosome-positive ALL (Ph+ ALL). Ph-like signature is presumably driven by kinase-activating gene alterations. Thus, both gene expression pattern and DNA mutational status should be assessed to make a definitive diagnosis for Ph-like ALL. A variety of approaches combining multiple methods, including RNA sequencing (RNA-seq), Taqman low-density array (LDA), fluorescence in situ hybridization (FISH) and targeted DNA sequencing, are being tested; however, such multi-omics approaches are available only in limited institutions. Since Ph-like ALL patients generally exhibit poor response to standard chemotherapy, and tyrosine kinase inhibitors (TKIs) may benefit them when used in a timely manner, a fast, accurate and generalizable diagnostic method is critically needed. In the present study, we have developed a nCounter-based diagnostic method for Ph-like ALL and validated it using a cohort of Japanese adult B-ALL cases. To identify genes that are uniquely expressed (or not expressed) in Ph+ B-ALL, we first obtained publicly-available gene expression datasets comprising 1146 B-ALL cases and identified 82 differentially-expressed genes in Ph+ ALL cases. We then assessed expression levels of those genes in an independent cohort using the nCounter, which enables fast, sensitive and accurate RNA detection. We also tested whether nCounter-based methods can detect fusion transcripts relevant for Ph-like ALL pathogenesis using probes targeting ABL1, ABL2, CSF1R, PDGFRB, and JAK2. We analyzed 123 samples (Ph+ = 42, Ph- = 81, age 16 to 67) obtained from newly-diagnosed adult B-ALL patients enrolled in two clinical trials conducted by the Fukuoka Blood and Marrow Transplantation Group (FBMTG) (Nagafuji et al. Eur J Haematol 2019). Unsupervised hierarchical clustering successfully stratified 123 cases into two disease clusters: Ph+ and Ph- subgroups. As expected, Ph+ subgroup included almost all Ph+ ALL cases (40 out of 42 cases), while 18 out of 81 Ph- ALL cases (22%) were categorized into the Ph+ subgroup. We defined these cases as Ph-like ALL. To validate the nCounter-based Ph-like ALL classification, we performed RNA-seq and target-capture DNA sequencing of all Ph- ALL cases. As expected, we detected kinase-activating fusions/rearrangements, including CRLF2 rearrangements (7 cases), PDGFRB fusions (3 cases), JAK2 fusions (2 cases), EPOR rearrangements (2 cases), ABL1 fusion (1 case), and FLT3 internal tandem duplication (1 case) in 16 Ph-like ALL cases, while no genetic alterations were detected in 2 cases. Fusion genes involving PDGFRB were consistently detected by nCounter (3/3); however, detection of those involving JAK2 (1/2) and ABL1 (0/1) were inconsistent. JAK2 and/or RAS mutations were detected in 5 of 7 Ph-like ALL cases harboring CRLF2 rearrangements. Of note, CRLF2 protein expression was detected by FACS in all CRLF2-rearranged cases. We next assessed significance of the Ph-like signature on clinical outcomes using a cohort of 40 Ph- ALL cases, in which minimal/measurable residual disease (MRD) status, assessed by IgH and/or TCR rearrangements, as well as clinical data were available (Nagafuji et al. Eur J Haematol 2019). Ph-like ALL cases exclusively exhibited MRD positivity after induction therapy as compared to non-Ph-like cases (p=0.04), indicative of the chemo-resistant nature of Ph-like ALL as previously reported (Roberts et al. N Engl J Med, 2014 and Roberts et al. J Clin Oncol, 2017). As expected, Ph-like ALL cases exhibited significantly poor disease-free survival compared with non-Ph-like ALL cases (p=0.04); however, no significant difference was evident in overall survival (p=0.62) presumably due to the fact that all MRD-positive cases were subjected to allo-HSCT after induction therapy. These data indicate that MRD-based therapy stratification could overcome chemo-resistant nature of Ph-like ALL. Our data suggest that nCounter-based diagnostic method is fast and accurate to identify Ph-like ALL. Since Ph-like signature generally dictates poor clinical outcomes, and upfront TKI therapy may improve them, our method could facilitate precision medicine in the treatment of Ph- B-ALL. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Targeting Activated Signaling Pathways for the Treatment of IKZF1-Deleted B Lymphoblastic Leukemia

In B lymphoblastic leukemia (B-ALL), genome-wide association studies have revealed that deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are present in nearly 30% of patients. These lesions are most prevalent in high-risk subsets, including greater than 60% of patients with Philadelphia chromosome positive (Ph+) and Ph-like ALL. IKZF1 deletions are associated with an increased risk of relapse, therapy resistance, and inferior survival. It is therefore imperative to devise new treatment strategies for this poor-prognosis subset of patients. To this regard, using novel CRISPR-Cas9 genome editing strategies, we developed a series of human B-ALL cell lines with IKZF1 deletions. These robust model systems have allowed us to investigate the underlying biology of IKZF1-deleted B-ALL. Our studies have thus far shown that IKZF1 deletion results in a stem cell-like gene expression profile, enhanced bone marrow homing and engraftment, and cell-intrinsic chemoresistance, consistent with the relapsing disease phenotype observed in affected patients. We are using these model systems to explore possible mechanisms of chemoresistance and delineate new strategies to improve response to therapy. Global gene expression analysis of the engineered Nalm-6 IKZF1-deleted cells by RNA-seq revealed potential therapeutic vulnerabilities. IKZF1-deleted cells are characterized by increased activation of the JAK/STAT pathway with overexpression of JAK1, JAK3, STAT3, and STAT5. Aberrant activation of this pro-survival, anti-apoptosis pathway is associated with poor-prognosis leukemia; thus, we postulated this is a contributor to the chemoresistance inherent to IKZF1-deleted B-ALL. We explored the therapeutic potential of targeting the JAK/STAT pathway by treating IKZF1-deleted cells with selective inhibitors of JAK1/3 (tofacitinib) and STAT3 (MM-206) and calculated the IC50 by Annexin V/7-AAD double-negative population after 48 to 72 hours of treatment. The IKZF1 wild-type cells were sensitive to both compounds, suggesting activated JAK/STAT signaling is critical to cell survival. In comparison, the IKZF1-deleted cells were relatively resistant to both compounds (MM-206 IC50 : 5.6 µM vs. 8.2 µM, p &lt; 0.001; tofacitinib IC50 : 43 nM vs. 55 nM, p = 0.05) similar to the relative resistance to ABL1-tyrosine kinase inhibition observed in Ph+ B-ALL cells with loss of function IKZF1 mutations. However, we postulated that inhibition of the JAK/STAT pathway could still augment the effects of standard chemotherapy. Indeed, whereas IKZF1-deleted Nalm-6 cells are highly resistant to glucocorticoid chemotherapy alone, when the cells were also treated with sub-IC50 levels of MM-206, we observed a significant re-sensitization to dexamethasone-induced apoptosis. A similar pattern of re-sensitization was seen with the combination of sub-IC50 MM-206 and vincristine treatment. Additionally, our gene expression analysis of the IKZF1-deleted Nalm-6 cells revealed significantly increased expression of the receptor tyrosine kinase, FLT3. Overexpression was confirmed at the protein level by flow cytometry for cell-surface FLT3. We treated our engineered cell lines with the potent and selective FLT3 inhibitor quizartinib and again found that the IKZF1-deleted cells were relatively resistant compared to the wild type cells (IC50 : 240 nM vs. 282 nM, p &lt; 0.01). Postulating that parallel activation of the JAK/STAT pathway may contribute to this resistance, we treated our cells with pacritinib, a combined JAK/FLT3 inhibitor. The IKZF1-deleted cells were as sensitive to this compound as the wild type cells, suggesting dual targeting of FLT3 and JAK may be efficacious for the treatment of IKZF1-deleted B-ALL. Our data support that IKZF1-deleted B-ALL is an aggressive disease characterized by cell-intrinsic chemoresistance. We found that loss of IKAROS amplifies pro-survival, anti-apoptotic signaling pathways, a likely contributing mechanism to chemoresistance. IKZF1 deletion confers relative resistance to targeted inhibitors of these pathways. However, the combined JAK/FLT3 inhibition exhibits therapeutic efficacy. Additionally, the combination of a JAK/STAT pathway inhibitor with conventional chemotherapy including dexamethasone and vincristine may be a promising strategy to overcome the chemoresistance inherent to this poor-prognosis subset of B-ALL. Disclosures No relevant conflicts of interest to declare.

Anti-CD19 chimeric antigen receptor T-cells induce durable remission in relapsed Philadelphia chromosome-positive ALL with T315I mutation

Effective treatments for relapsed Ph+ALL with T315I mutation are few; CD19 CAR T-cell therapy are a potential therapy for this condition. We reported 7 patients with relapsed Ph+ALL with T315I mutation, who were treated pre- or post-allo-HSCT with CD19-specific CAR T-cells. Of the 7 cases, 6 were in CR or CRp within 1 month after the first infusion of CAR T-cells. MRD revealed a rapid decline in 6 patients. BCR/ABL fusion transcripts were negative in 4/5 cases (not performed in 2). Three patients maintained remission without evidence of MRD by QPCR until the final follow-up, of which 2 received anti-CD19 CAR T-cells and ponatinib at the same time. Our study confirmed the efficacy of anti-CD19 CAR T-cell therapy in treatment of relapsed Ph+ALL with T315I mutation pre- or post-allo-HSCT and the concurrent applicability of this therapy with ponatinib.

SLC2A5 Overexpression in Childhood Philadelphia Chromosome Positive Acute Lymphoblastic Leukaemia

To study glycolysis/glycogenesis-related genes expression in childhood B cell acute lymphoblastic leukaemia (B-ALL), we performed a microarray-based analysis using published gene expression profiles. We found that gene SLC2A5, which encoded fructose transporter GLUT5 that facilitated cell fructose uptake, was up-regulated in Philadelphia chromosome positive ALL (Ph+ALL). Microarray-based analyses also suggested that SLC2A5 expression was significantly down-regulated in childhood B-ALL with t(1;19) or 11q23 mutation. High SLC2A5 expression was found in the patients who had recurrence within 3 years, early relapse, shortened complete remission duration, and positive minimal residue disease (MRD) status after treatment. The overexpression of SLC2A5 at both mRNA level and protein level in Ph+ALL was confirmed in a validation cohort of childhood B-ALL. We also validated the correlation of SLC2A5 expression and MRD status. In a mechanistic study using a human Ph+ALL cell line, we found that BCR-ABL kinase might regulate GLUT5 expression via c-myc. The tyrosine kinase inhibitors imatinib and dasatinib repressed GLUT5 expression and the cell uptake of fructose. Fructose protected the tumour cells from nutrition deficiency and drug-induced cell death. Overall, our findings showed that SLC2A5 was up-regulated in childhood Ph+ALL. The expression of SLC2A5 correlated with childhood B-ALL clinical factors, such as MRD status. Since TKI was able to inhibit GLUT5 expression, repression of fructose utility after TKI treatment contributes to TKI-induced Ph+ALL cytotoxicity. Targeting GLUT5 might be promising in B-ALL treatment, especially for Ph+ALL patients with high expression of SLC2A5. DisclosuresNo relevant conflicts of interest to declare.

Ruxolitinib in Combination with Dasatinib and Dexamethasone Is an Active and Well-Tolerated Chemotherapy-Sparing Oral Induction Regimen for Adults with Philadelphia Chromosome-Positive ALL: Results of a Phase I Trial

Introduction: Induction therapy consisting of dasatinib (DAS) and corticosteroids (CS) yields high rates of morphologic complete response (CR) in adults w/ newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). However, only ~20% of patients (pts) achieve BCR-ABL1 response depth of Methods: We conducted a phase I trial (NCT02494882) w/ accelerated dose escalation of RUX. Pts w/ newly diagnosed Ph+ ALL received CS pre-phase (d -6 to 0) followed by DAS 140 mg daily (starting d1) in combination w/ dexamethasone (DEX) 10 mg/m2 PO daily (d1-24, then taper) and RUX (d1-84). RUX doses were 10 mg PO BID (n=1), 15 mg PO BID (n=1), 20 mg PO BID (n=1), and 25 mg PO BID (n=7). IT methotrexate was given as CNS prophylaxis (d22, 43, 64, 85). Post-induction therapy was left to the discretion of the treating physician. The primary objective was to determine the recommended phase 2 dose (RP2D) of RUX in this combination. Secondary objectives included assessment of response kinetics, including minimal residual disease (MRD) negativity by flow cytometry (FACS) and BCR-ABL1 transcript monitoring, and ABL kinase mutation development. Results: Ten pts enrolled (6M, 4F). Median age at time of screening bone marrow (BM) studies was 63.8 yrs (range, 25.7-77.4; 7/10 age ≥60). BCR-ABL1 transcripts were p190 (n=7), p210 (n=2), atypical (n=1). No dose-limiting toxicity (DLT) has been observed. Enrollment was expanded at the highest RUX dose level (25 mg PO BID). Grade (Gr) 3-4 non-hematologic toxicities possibly/probably attributable to DAS/RUX/CS were limited to Gr 3 hyperglycemia in the setting of CS (n=5), Gr 3 hypofibrinogenemia (n=1), and Gr 3 peripheral edema (n=1). Two pts discontinued DAS after completing the 84d induction due to symptomatic pleural effusions. All pts achieved morphologic CR or CR w/ incomplete hematologic recovery (CRi). Median log reduction in BCR-ABL1 transcript levels (Figure 1A) was 2.7 at d22 (range, 1.3-4.2, n=9), 3.4 at d43 (range, 1.0-CMR, n=8), and CMR at d85 (range, 3.2-CMR, n=5). By FACS, 7/10 achieved MRD Among 6 pts completing the 84d induction, next post-remission therapy included tyrosine kinase inhibitor (TKI) + concurrent blinatumomab (n=3), TKI + hyperCVAD (n=1), and allogeneic hematopoietic cell transplantation (alloHCT, n=1). Of the 4 pts not completing the 84d induction, next therapy included ponatinib + blinatumomab (n=2), ponatinib + hyperCVAD (n=1), or DAS alone (n=1). Overall, 3 pts experienced morphologic relapse, which was observed in the setting of T315I (n=2) or F317L (n=1) ABL kinase mutations. Median EFS and OS (Figure 1B&C) are not yet reached in this small group of pts at median follow-up of 11.2 months among survivors (range, 2.8-20.7). Conclusions: Chemotherapy sparing-induction consisting of DAS, RUX, and initial DEX was well tolerated among adults w/ newly-diagnosed Ph+ ALL and led to CR/CRi in all pts. The RP2D of RUX is 25 mg PO BID. Kinetics of BCR-ABL1 transcript reduction compare favorably w/ other series of TKI + steroid-based induction, w/ rapid achievement of median >3 log reduction in BCR-ABL1 transcripts. Correlation of response kinetics w/ JAK/STAT pathway inhibition may suggest mechanisms of resistance apart from ABL kinase mutations. In future chemotherapy-sparing induction approaches, incorporating ponatinib, asciminib, or other novel therapies (vs. DAS) during induction may suppress leukemic clones bearing T315I mutations and reduce early relapse rates. Disclosures Geyer:Dava Oncology: Honoraria. King:Genentech: Other: Advisory Board . Mauro:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Takeda: Consultancy. Tallman:BioSight: Other: Advisory board; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Daiichi-Sankyo: Other: Advisory board; Orsenix: Other: Advisory board. Levine:Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership; Novartis: Consultancy; C4 Therapeutics: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Prelude: Research Funding; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Imago: Equity Ownership. Douer:Jazz Pharmaceuticals: Consultancy; Gilead Sciences: Consultancy; Shire: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria; Spectrum: Consultancy. Park:Novartis: Consultancy; Kite Pharma: Consultancy; Adaptive Biotechnologies: Consultancy; AstraZeneca: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Amgen: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Pfizer: Consultancy; Shire: Consultancy.

Phase II Study of Blinatumomab in Patients with B-Cell Lineage Acute Lymphocytic Leukemia with Positive Minimal/Measurable Residual Disease

Phase II Study of Blinatumomab in Patients with B-Cell Lineage Acute Lymphocytic Leukemia with Positive Minimal/Measurable Residual Disease

SLC2A5 overexpression in childhood philadelphia chromosome-positive acute lymphoblastic leukaemia.

To study glycolysis/glycogenesis-related genes expression in childhood B-cell acute lymphoblastic leukaemia (B-ALL), we performed a microarray-based analysis using published gene expression profiles. We found that SLC2A5, which encodes solute carrier family 2 member 5 (SLC2A5, previously termed GLUT5) that facilitates cell fructose uptake, was up-regulated in Philadelphia chromosome-positive ALL (Ph+ALL). Microarray-based analyses also suggested that SLC2A5 expression was significantly down-regulated in childhood B-ALL with t(1;19) or 11q23 mutation. High SLC2A5 expression was found in patients who had disease recurrence within 3years, early relapse, shortened complete remission duration and positive minimal residue disease (MRD) status after treatment. SLC2A5 overexpression at both the mRNA and protein level in Ph+ALL was confirmed in a validation cohort of childhood B-ALL. We also validated the correlation of SLC2A5 expression and MRD status. A mechanistic study using a human Ph+ALL cell line showed that BCR-ABL1 kinase might regulate SLC2A5 expression via MYC. The tyrosine kinase inhibitors (TKIs) imatinib and dasatinib repressed SLC2A5 expression and the cell uptake of fructose. Fructose protected the tumour cells from nutrition deficiency and drug-induced cell death. Overall, our findings showed that SLC2A5 was up-regulated in childhood Ph+ALL. SLC2A5 expression correlated with childhood B-ALL clinical factors, such as MRD status. Given that TKIs could inhibit SLC2A5 expression, repression of fructose utility after TKI treatment contributes to TKI-induced Ph+ALL cytotoxicity. Targeting SLC2A5 might be promising in B-ALL treatment, especially for Ph+ALL patients with high SLC2A5 expression.

Ponatinib evaluation and safety in real-life chronic myelogenous leukemia patients failing more than two tyrosine kinase inhibitors: the PEARL observational study

Ponatinib represents a remarkable progress in the treatment of heavily pretreated chronic myelogenous leukemia (CML) and de novo Philadelphia chromosome-positive ALL patients despite significant toxicity in clinical trials. To date, "real-life" data remain few and the use of ponatinib in this setting and its consequences remain mostly unknown. We report, within a national observational study, the use of ponatinib in unselected CML patients who had previously failed ≥2 lines of tyrosine kinase inhibitor (TKI) therapy (or one line if an Abelson (ABL)T315I mutation was identified), in real-life conditions (2013-2014) in a compassionate program. Our analysis has been focused on 48 chronic phase CML patients recorded. With a median follow-up of 26.5 months since ponatinib initiation, the overall survival (OS) rates (80.5% at 3 years) and cumulative incidence of major molecular response (81.8% at 18 months) were similar to those of the phase II study, with no influence of BCR-ABL mutations nor the reason of ponatinib prescription. A specific subanalysis of the preexisting cardiovascular risk factors and events occurring on ponatinib is described. These events occurred after a median time on ponatinib of 5.8 months (excluding hypertension) and were observed in 29/48 patients (47%), even in those already on anti-aggregants/coagulants. The majority were not severe and resolved, but two cases were fatal. Other hematological or nonhematological nonvascular adverse events were similar to those previously described in trials. This observational study reports similar rates of survival, molecular responses, and a slight increase in the cardiovascular toxicity of ponatinib in real-life conditions, prompting improved control of cardiovascular risk factors and selection of patients.

Current management of Philadelphia chromosome positive ALL and the role of stem cell transplantation.

Treatment of Philadelphia chromosome positive acute lymphoblastic leukemia exemplifies how the addition of potent targeted agents, directed at the molecular aberrations responsible for leukemic transformation, can overcome resistance mechanisms to traditional regimens and lead to improved outcomes. The introduction of BCR-ABL1 targeted tyrosine kinase inhibitors (TKIs) has significantly improved the outcomes not only by allowing more patients to undergo allogeneic hematopoietic cell transplantation (alloHCT) but also by decreasing our reliance on this potentially toxic strategy, particularly in the less fit population. Long-term data using chemotherapy and TKI combinations demonstrate that a proportion of patients treated can achieve durable relapse-free survival without undergoing alloHCT. Furthermore, the availability of sensitive minimal residual disease monitoring assays may allow early detection of the patients who are more likely to relapse and who are likely candidates for early alloHCT. The emergence of more potent TKIs with significant activity against resistant mutations has allowed deintensification of chemotherapy regimens. Available data indicate that complete reliance on TKIs, alone or with minimal additional therapy, and elimination of more intensive chemotherapy or alloHCT is unlikely to achieve long term cure in most patients. However, introduction of other highly effective agents that can be combined with TKIs may allow further minimization of chemotherapy and alloHCT in the future, as we have witnessed in acute promyelocytic leukemia.

Phase II study of hyper-CMAD with Liposomal Vincristine (Marqibo) for Patients with Newly Diagnosed Acute Lymphoblastic Leukemia

Abstract Background: Liposomal vincristine is designed to reduce neurotoxicity and increase dose intensity delivery, and has been approved as salvage therapy for patients with relapsed / refractory ALL. Replacement of regular vincristine with liposomal vincristine might lead to improve response rate and survival as well as reduced toxicity. Methods: Patients ≥18 years with newly-diagnosed B-cell ALL were eligible for the phase II clinical trial consisting of hyper-CMAD (cyclophosphamide 300 mg/m2 IV every 12 hours on days 1-3; liposomal vincristine 2.0 mg/m2 IV on day 1 and day 8; doxorubicin 50 mg/m2 IV on day 4; dexamethasone 40 mg IV daily on days 1-4 and days 11-14) (odd cycles 1, 3, 5, 7) alternating with high-dose methotrexate 1000 mg/m2 IV on day 1, and cytarabine 3 gm/m2 IV every 12 hours on days 2, 3 (even cycles 2, 4, 6, 8). Rituximab 375 mg/m2 IV on days 1 and 8 for courses 1-4 was administered in CD-20 positive ALL. Imatinib 600 mg PO daily or dasatinib 100 mg PO daily days 1-14 on course 1 and continuously (imatinib 600 mg PO or dasatinib 70 mg PO) daily starting day 1 after the first cycle in Philadelphia chromosome positive (Ph+) ALL. Results: Thirty-one patients (16 men, 15 women) have been treated. Patient characteristic and response are described in Table 1. Median follow-up is 33 months (5-46) with a median cycle of 4 cycles (1-8). Median age is 53 years (range 19-80). Thirteen patients (42%) had CD-20 positive ALL, and 21 (68%) had Ph+ ALL. Thirty (97%) achieved complete remission (CR). Early death was observed in 1 patient (3%) with Ph+ ALL. Of 26 patients evaluable for response, 26 (100%) achieved complete cytogenetic response (3 patients, diploid at start; 1 patient, not performed), and 27 (90%) achieved negative minimal residual disease status by multicolor flow cytometry. Of 20 evaluable patients with Ph+ ALL, major molecular response was achieved in 19 patients (95%); complete molecular response in 14 (70%). Grade 3/4 toxicity ≥10% included infections in 28 (90%); hyperglycemia, 16 (52%); hypokalemia, 15 (48%); hypophosphatemia, 15 (45%); hypocalcemia, 10 (32%); DVT, 7 (23%); hyponatremia, 7 (23%); hypoalbuminemia, 6 (19%); peripheral neuropathy, 5 (16%); elevated ALT, 5 (16%); and acute kidney injury 4 (13%). All grade peripheral neuropathy was observed in 21 (68%). With a median follow-up of 33 months, 21 (68%) patients are alive. Ten (32%) patients died: 1, sepsis on C1D10; 4, unknown; 1, post-transplant complications; 4, relapse. The 2-year CR duration and survival rates were 83% and 68%, respectively (Figure 1). Conclusions: Hyper-CMAD with liposomal vincristine is safe and shows high response and survival rates in patients with newly diagnosed ALL. Download : Download high-res image (147KB) Download : Download full-size image Disclosures Jabbour: Bristol-Myers Squibb: Consultancy. Thomas: Amgen: Honoraria; Pfizer: Honoraria. Cortes: BMS: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Sun Pharma: Research Funding; ARIAD: Consultancy, Research Funding; Teva: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. O'Brien: Janssen: Consultancy; Sunesis: Consultancy; Regeneron: Other: Research Support: Honorarium, Research Funding; AbbVie: Consultancy; Celgene: Consultancy; CLL Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Acerta: Other: Research Support: Honorarium, Research Funding; Aptose Biosciences, Inc.: Consultancy; Amgen: Consultancy; Pharmacyclics: Consultancy, Other: Research Support: Honorarium, Research Funding; Alexion: Consultancy; Astellas: Consultancy; GSK: Consultancy; TG Therapeutics: Consultancy, Other: Research Support: Honorarium, Research Funding; ProNAI: Other: Research Support: Honorarium, Research Funding; Gilead Sciences, Inc.: Consultancy, Other: Research Support: Honorarium, Research Funding; Vaniam Group LLC: Consultancy; Pfizer: Consultancy, Research Funding. Kantarjian: Amgen: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding.

Abstract 5829: Targeting TSLP-induce upregulation of Mcl-1 for the treatment of Ph-like ALL with CRLF2 alterations

Abstract B cell precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. The subset of pediatric B-ALL patients at greatest risk for relapse and death have a gene expression profile similar to Philadelphia chromosome positive ALL. Approximately half of these Ph-like B-ALL are defined by genetic alterations that result in overexpression of the cytokine receptor, CRLF2. Stimulation of the CRLF2 receptor by the cytokine, TSLP, causes downstream activation of the JAK/STAT5 and PI3/AKT/MTOR pathways. Activation of these pathways has been associated with oncogenesis and chemoresistance. A gene target of activated STAT5 in B cell precursors is Mcl-1, a Bcl2 family pro-survival molecule. Further, Mcl-1 protein levels are increased through post-transcriptional mechanisms induced by activation of the mTOR pathway. We hypothesized that circulating TSLP cytokine contributes to chemoresistance by increasing CRLF2 activation leading to increased Mcl-1 expression and that targeting Mcl-1 could be an effective strategy for treating Ph-like B-ALL with overexpression of CRLF2 (CRLF2 B-ALL). To test this hypothesis we cultured human CRLF2 B-ALL cell lines (MUTZ5 and CALL4) with and without TSLP for 3 days and evaluated expression of Mcl-1 by flow cytometry. We found that TSLP induced significant increases in Mcl-1 proteins in both cell lines. To determine if these results are reflective of what happens in patients, primary CRLF2 B-ALL cells from pediatric patients were cultured with physiological levels of TSLP (~20 pg/ml) and similarly evaluated. Physiological TSLP significantly increased Mcl-1 protein in primary CRLF2 B-ALL cells, including those with activating JAK mutations. Our next question was whether TSLP-induced increases in Mcl-1 could be effectively targeted with Mcl-1 inhibitors (MIM-1 or Maritcolax). CRLF2 B-ALL cells were incubated with and without TSLP and treated with increasing doses of Mcl-1 inhibitor. Dose responses were evaluated by flow cytometry after 2 or 3 days. Mcl-1 inhibitors induced dose-dependent decreases in cell count and increases in caspase-3 activation and apoptosis (Annexin V/7-AAD). These corresponded with dose-dependent decreases in Mcl-1 protein, suggesting that both inhibitors target Mcl-1 for degradation. MIM-1 and Maritoclax showed efficacy against both CRLF2 B-ALL cell lines and primary patient samples, including those with activating JAK mutations, although cells cultured with TSLP typically required twice the dose of Mcl-1 inhibitor to achieve the same effect observed without TSLP. These data provide evidence that TSLP can contribute to leukemia cell survival and identify Mcl-1 inhibitor as a candidate therapy for CRLF2 B-ALL. Ongoing studies are evaluating the efficacy of the Mcl-1 inhibitor, Maritoclax, in novel patient-derived xenograft models of CRLF2 B-ALL that provide physiological levels of human TSLP. Citation Format: Cornelia Stoian, Nathaniel George Mambo, Pierce McCarthy, Veriah Vidales, Jacqueline S. Coats, Ineavely Baez, Sinisa Dovat, Shadi Farzin Gohar, Dhimant Desai, Muhammad Kamal, Kimberly J. Payne. Targeting TSLP-induce upregulation of Mcl-1 for the treatment of Ph-like ALL with CRLF2 alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5829. doi:10.1158/1538-7445.AM2017-5829

New Treatment Strategies for Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia.

To review recent studies that address important questions regarding the treatment of Philadelphia chromosome-positive ALL. Less intensive non-myelosuppressive induction approaches can produce comparable anti-leukemic responses with less toxicity. Second-generation tyrosine kinase inhibitors (TKIs) are not clearly associated with superior outcomes compared to imatinib. Ponatinib is associated with lower early relapse rates, but has additional vascular risks. Patients achieving complete molecular response (CMR) appear to have comparable survivals with chemotherapy + TKI alone as compared with allogeneic stem cell transplant (alloSCT). In contrast, those not achieving CMR have superior outcomes with alloSCT. There is no clear evidence of benefit for post-transplant TKI therapy. Patients resistant to chemotherapy + TKIs may respond to blinatumomab or inotuzumab; these agents may also convert some patients to CMR. Future studies should further explore the potential of ponatinib and antibody-based therapies to more precisely define optimal molecular responses.

Lenalidomide Effectively Induces Philadelphia-Chromosome Positive ALL Cells into Apoptosis in Synergy with Tyrosine Kinase Inhibitor

[Background] Lenalidomide (LMD) has been effectively used for the treatment of hematologic malignancies such as multiple myeloma and 5q-MDS. Recently, LMD was shown to bind the celebron (CRBN) E3 ubiquitin ligase complex and degrade Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3) via ubiquitin-proteasome system in myeloma cells, which leads to decrease in expression of downstream targets such as IRF4 and c-Myc. The IKZF1 is an essential transcription factor for the development of all lymphoid lineages particularly in the development of B- cell progenitors (BCP), and its gene consists of 8 exons including exons 4-6 and exon 8 encoding DNA-binding and dimerization domains, respectively. Genomic heterozygous deletions in IKZF1 are found in ~15% childhood BCP-ALL and the most frequent deletions affect the whole gene or exons 4-7 resulting in haploinsufficiency or expression of a dominant negative isoform. The IKZF1 deletions are now a hallmark for refractory nature of BCP-ALL linking to an unfavorable clinical outcome, and the frequency of IKZF1deletion is remarkably high (~70%) in Philadelphia-chromosome positive (Ph+) ALL cases. It is thus of great interest to examine LMD's effects on Ph+ALL cells.[Materials mad Methods] A total of 7 Ph+ALL cell lines with IKZF1 deletions (4 in exons 4-7, 2 in exons 2-7, and 1 in exons 1-8) were used to examine LMD's effects. The synergic effects of LMD with tyrosine kinase inhibitor (TKI; imatinib, dasatinib) were also examined. The ex vivo studies using NOD/Shi-scid, IL-2Rgnull (NOG) mice model were performed.[Results] 1. We first performed [3H]-thymidine uptake assays in the presence or absence of 20µM LMD for 3-6 days, and found that thymidine uptakes were variably inhibited (8~65%) after LMD treatment and two of sensitive cell lines showed a marked inhibition in a dose and a time dependent manners of LMD (~75%). Of importance, their thymidine uputakes were further inhibited (~95%) by LMD in the presence of the imatinib in a dose dependent manner (0.05-0.5nM). 2. To access the inhibition of thymidine uptake, we performed alamarBlue cytotoxic assays using the sensiitive cell line KOPN57bi, and found that the cell numbers modestly decreased by LMD (20 µM) or imatinib (0.5nM) , and markedly decreased (up to 35% at day 4) by LMD+imatinib. In flow cytometric analysis, KOPN57bi treated with LMD or imatinib showed cell cycle arrest alone, while the cell line treated with LMD+imatinib showed a marked increase in the subdiploid (42.3%), Annexin V-positive (72.0%), and active caspase 3-positive (31.8%) populations at day 4 after trreatment. The pancaspase inhibitor Z-VAD-FMK completely inhibited induction of cell death (apoptosis) by LMD+imatinib. These results were also obtained by use of another TKI dasatinib instead of imatinib. 3. We examined changes in expression of IKZF1 on Western blot using anti-C-terminal IKZF1 antibody in KOPN57bi (deleted in exons 4-7) 24h after LMD and/or TKI treatment, and found that several alternative splicing forms translated from intact IKZF1 disappear by LMD, but expression of a dominant negative Ik6 isoform was not affected by LMD presumably due to a lack of the CRBN binding site (residue Gln 146:exon5) in Ik6 isoform. IRF4 was also down-regulated by LMD and further in the presence of TKI. 4. We performed ex vivo studies using NOG mice. After intravenous injection of KOPN57bi (2x106cells), mice were orally administrated with saline (control, n=4), LMD (30mg/kg/d, n=5), imatinib (150mg/kg/d, n=5) , or LMD+imatinib (n=4) for 10 days (day 0,1,2,3,4, 7,8, 9,10,and 11) and percentages of human CD45 positive population in bone marrow mononuclear cells were examined at day 14. Human CD45+ cells showed 29.7±4.3% (mean±SEM) and 30.3±1.5% in control and imatinib-treated mice, respectively, whereas they were significantly (p=0.0002) decreased to 2.1±0.4% in LMD-treated mice. Of importance, they were further decreased to 0.3±0.4% in LMD+imatinib treated mice (p=0.0051), suggesting that combined treatment of LMD and imatinib should be very effective in xenograft mice models.[Conclusion] LMD renderd Ph+ALL cells a marked cell cycle arrest by completely abrogating the remaining IKZF1 function, and effectively induced them into apoptosis in synergy with TKI. Orally administration of LMD and TKI might become a high QOL treatment option for the treatment of Ph+ALL patients particularly for those with a higher age and organ dysfunctions. DisclosuresNo relevant conflicts of interest to declare.