Lymphoid Blast Crisis Research Articles

B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Specific Copy Number Alterations Are Unique For Progressive Pediatric Chronic Myeloid Leukemia (CML): A Large Cohort Study

Chronic myeloid leukemia (CML) is a rare malignancy in children and is mostly diagnosed in the chronic phase (CP). In adults, the five-year overall survival rate is 89% for patients on Imatinib and disease progression occurs in 1-3% per year (Druker 2006). Once a blast crisis (BC) has occurred, treatment options are limited with a median survival of only a few months (Cortes 2008). Therefore, early recognition of patients at risk for developing a BC is desirable. Besides the translocation t(9;22)(q34;q11), IKZF1, PAX5, and CDKN2A deletions have been reported in CML lymphoid blast crisis (LyBC) of both adult and pediatric patients (Mullighan 2008, Alpár 2012).The aim of this study was to investigate the presence of IKZF1 deletions and other copy number alterations (CNAs) by MLPA analysis in a large cohort of pediatric CML patients at time of diagnosis in order to determine whether CNAs commonly found in pediatric ALL might predict disease progression and / or treatment response.Between October 1991 and October 2012 a total of 86 children with newly diagnosed CML were included. The median follow up was 31 months.Among the 86 patients, 82 patients were diagnosed in CP, 2 patients in accelerated phase (AP), and 2 patients in LyBC. Six patients experienced progression to a BC respectively a myeloid blast crisis (MyBC) (N=2) and LyBC (N=4).At time of diagnosis, an IKZF1 deletion was detected in one patient diagnosed with CML-AP (Table A, patient no 58). IKZF1 and EBF1 deletions were detected in one patient diagnosed with CML-LyBC (Table A, patient no 22). No CNAs were detected in the 82 patients diagnosed with CML-CP. [Display omitted] At time of disease progression, new CNAs were detected at time of the LyBC (Table A, patient no 62, 64, and 67). Due to the absence of material no CNAs could be detected in both patients experiencing a MyBC.In conclusion, we were able to detect CNAs in progressive CML disease (CML-AP and CML-LyBC) and not in the samples at the time of chronic phase in this large pediatric cohort of CML patients. Therefore, the investigated CNAs could not be used to predict disease progression at time of diagnosis. The CNAs detected in patients with progressive CML were similar to specific CNAs detected in pediatric B-cell precursor ALL, indicating a similar disease development (Kuiper 2010).Additionally, our results are in accordance with existing literature, suggesting that mechanisms of disease progression in pediatric and adult CML might be similar (Brazma, 2007). Disclosures:No relevant conflicts of interest to declare.

Post-Transplant Cyclophosphamide Haplo-HSCT Revised: Peripheral Blood Stem Cell Graft and Sirolimus To Enhance Immune Reconstitution and Graft Versus Leukemia Effect In Patients With Active Leukemia

IntroductionAchieving a cure for relapsed leukemia remains a challenge. Hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative provides a potentially curative treatment for such patients in urgent need. Based on previous studies on the feasibility and low toxicity profile of haploidentical HSCT with high-dose post-transplant Cyclophosphamide (PT-Cy), we planned to intensify this protocol for patients with active disease by the use of: 1) myeloablative conditioning, 2) peripheral blood stem cells (PBSCs) graft instead of bone marrow (BM), and 3) sirolimus instead of tacrolimus, for its direct anti-leukemic effect. Patients and MethodsHere we report the results on 18 patients treated between November 2012 and July 2013. The median age was 59 years (range, 30–78). Diagnoses included AML (10 refractory, 3 CR1, and 2 CR>1), ALL (2 refractory), and 1 CML in lymphoid blast crisis. The majority (78%) of patients were not in remission at the time of HSCT. Consistently, 13 (72%) patients scored high/very high accordingly to the CIBMTR disease score (Todisco et al., Leukemia 2013). Fourteen (78%) patients had a Sorror comorbidity score ≥ 3. Conditioning consisted of treosulfan (14 g/m2/day) on days –6 to –4, fludarabine (30 mg/m2/day) on days –6 to –2, and melphalan (70 mg/m2/day) on days –2 and –1, followed by T-repleted peripheral blood stem cells (PBSCs). Postgrafting immunosuppression consisted of PT-Cy (50 mg/kg/day) on days 3 and 4, followed by mycophenolate mofetil for 30 days, and sirolimus for 3 months. ResultsPBSC grafts contained an infused median TNC/kg of 10.5 e8 (range 5.5–20.7 e8), CD3/kg 18.4 e7 (range 10.5–40.9 e7) and a CD34/kg of 7.01 e6 (range 4.3–8.02 e6). All patients but 1 engrafted (94%), with a median time to neutrophil and platelet recovery of 18 days (range 14–26) and 19 days (range 9–51) respectively. Notably, the patient with primary graft failure had a HLA-DP mismatch in HvG direction. No patient experienced secondary graft failure. Post-HSCT recovery of lymphocyte subsets was broad and fast: the median CD3 count at day 30 after HSCT was 642/µl and exceeded 1000/µl by day 60, with a median value of CD4 T cell counts of 329/µl at day 30; NK cells reached a median value of 192/µl at day 30 and remained stable thereafter, while B cells steadily increased from a median value of 2/µl at day 30 to 48/µl at day 180. Circulating T cells comprised both naïve and memory subsets, with a recovery of CD31+ recent thymic emigrants (RTEs) starting from day 30. All patients had a significantly higher proportion of circulating RTEs at day 30, 90 and 180 compared to their pre-HSCT levels, suggesting an improvement in their thymic function after HSCT. Peri-engraftment syndrome (PES) occurred in 4 patients with a median time to onset of 15 days and was promptly resolved with short-course steroids. Interestingly, the percentage of circulating Ki-67+, dividing, effector memory CD8 T cells at day 8 post HSCT was able to accurately predict the occurrence of PES. Grade I/II skin acute GvHD occurred in 6 patients, while 4 patients experienced grade III/IV acute GvHD (22%). Acute GvHD were accompanied by a rise in circulating Ki-67+ CD8 T cells, while response to first line therapy resulted in a drop in Ki-67 expression. Chronic GVHD occurred in 2 of the 7 patients at risk, but only one patient required systemic therapy. Of the 16 patients at high risk, 7 had CMV reactivations. No patient had EBV-related PTLD. With a median follow-up of 100 days (range 15–260), 14 (78%) out of the 18 patients are alive. Only two patients died of non-relapse mortality (NRM, 11%). All patients in CR at time of transplant are alive and disease-free, while 8 (57%) out of 14 patients with active disease at transplant are alive and leukemia-free. In 4 patients tumor-specific T cells against PRAME or WT1 were detectable both in the peripheral blood and BM up to 180 days after HSCT, suggesting that PT-Cy did not hamper the GvL effect. ConclusionsMyeloablative haploidentical HSCT with PBSCs, PT-Cy and sirolimus is feasible and might be a valid option for patients with refractory/active leukemia. If confirmed in a larger cohort of patients and with a longer follow-up, these results suggest that the acceptable rates of GvHD and NRM as well as the favorable immune reconstitution profile open the way for combining it with novel immunomodulatory or cellular therapies to improve leukemia-free survival. Disclosures:Bonini:MolMed SpA: Consultancy.

High Content Screening Identifies Synthetic Lethality Of Retinoid Receptor Agonists In IKZF1-Mutated BCR-ABL1 positive Acute Lymphoblastic Leukemia

Expression of BCR-ABL1 is a hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). While CML is typically responsive to tyrosine kinase inhibitor monotherapy, BCR-ABL1-positive ALL is associated with a high frequency of treatment failure and relapse, even in the era of TKI therapy. Alterations of the lymphoid transcription factor gene IKZF1 (IKAROS) are frequent in BCR-ABL1 ALL and in CML at lymphoid blast crisis. Moreover, IKZF1 alterations are associated with poor outcome in BCR-ABL1-positive and -negative B-ALL. IKZF1 alterations are usually heterozygous and include loss-of-function deletions (∼60% of cases), focal deletion resulting in expression of a dominant negative isoform lacking the N-terminal DNA-binding zinc fingers, IK6 (∼30%) and missense mutations (∼10%). In addition, over half of BCR-ABL1-positive ALL cases harbor deletions of CDKN2A/B (INK4/ARF).We used two murine models of BCR-ABL1 leukemia to examine the role of IKZF1 alterations (deletions and IK6) and ARF loss in disease lineage and treatment responsiveness. In the first model, lineage negative Arf-/-, Ikzf1+/- or wild type (WT) bone marrow was transduced with p185 BCR-ABL1-expressing retrovirus and either an IK6-expressing retrovirus or empty vector followed by transplantation into irradiated wild type mice. p185-transduced WT marrow invariably induced a CML-like leukemia, whereas Arf loss and Ikzf1 alterations synergistically drove an aggressive pre-B ALL. In a complementary model, unmanipulated Arf-/-, Ikzf1+/-, or WT marrow was transduced with p185 BCR-ABL and transplanted immediately or after in vitro derivation of pre-B cell cultures. In this model, Arf loss and Ikzf1 haploinsuffiency or expression of IK6 reduced the latency of ALL and reduced responsiveness to dasatinib in vitro and in vivo. Strikingly, perturbation of Ikzf1 activity by haploinsufficiency, expression of IK6, zinc finger missense mutations, or shRNA mediated knockdown was associated with increased expression of the stem cell marker CD90 (Thy1) and the growth of cells in large aggregates in liquid cultures. Ikzf1 alteration was also associated with induction of a hematopoietic stem cell-like gene expression program, and increased self-renewal manifested as enhanced serial colony forming potential. This aberrant growth and self-renewal phenotype was abrogated by neutralizing anti-Thy1 antibodies. Thy1 is a known IKZF1 target, and these findings suggest that IKZF1 alterations derepress Thy1 expression resulting in enhanced stemness and treatment resistance.To identify agents that may enhance TKI responsiveness, we screened the activity of 384 FDA approved compounds in Arf-/- BCR-ABL1+ IK6+ pre-B. We assessed cell viability using CellTiter Glo, and measured the number and size of cell aggregates by high throughput microscopy to identify drugs that selectively inhibit cell aggregation. This identified the retinoid X receptor agonist bexarotene as a potent inhibitor of cell aggregation. A follow-up screen of 128 nuclear hormone receptor effector compounds identified potent anti-aggregation activity of 9-cis retinoic acid, 13-cis retinoic acid, all-trans retinoic acid and carbacyclin. In contrast, N-oleoylethanolamine, an antagonist of perioxisome proliferator-activated receptors that heterodimerize with RXRs, markedly increased cell aggregation. All retinoid receptor agonists induced cell cycle arrest and reduced proliferation without inducing apoptosis, and attenuated Thy1 expression, abrogated colony formation and enhanced the efficacy of dasatinib in vitro and in mice transplanted with Arf-/- p185 IK6 cells. Notably, Ikzf1is a predicted retinoid receptor target, and bexarotene treatment upregulated expression and nuclear localization of wild type IKZF1 in Arf-/- p185 pre-B cells, with and without IK6.Thus, IKZF1 alterations drive lymphoid lineage, and induce self-renewal and drug resistance in BCR-ABL1 ALL. IKZF1 alterations are associated with overexpression of Thy1, a target of IKZF1 transcriptional repression. This phenotype is reversed by retinoid receptor agonists that induce expression of IKZF1, attenuate Thy1 expression and increase sensitivity to TKI therapy. These data provide important new insights into the role of IKZF1 alterations in leukemogenesis, and identify a new therapeutic option in high risk B-ALL. Disclosures:No relevant conflicts of interest to declare.

Mouse Versus Human Extrinsic Cues Dictate Transformation Potential In BCR-ABL/BMI1-Induced Leukemia In Humanized Xenograft Models

The NSG [NOD/Lt-scid/IL2Rγnull] xenotransplantation mouse model is currently the model of choice to evaluate human hematopoietic engraftment and to study development of human leukemia. Indeed, we have previously shown that co-expression of BCR-ABL together with the polycomb repression complex 1 (PRC1) member BMI1 in human cord blood (CB) derived CD34+ cells was sufficient to induce a serially transplantable lymphoid leukemia (Rizo et al., Blood 2010). This leukemia was characterized by high levels of CD34+/CD19+/CD20-/IgM-/CD33-/CD15- lymphoid blasts in the bone marrow and a high degree of infiltration of blasts in spleen and liver. Clonal analysis revealed that similar clones gave rise to leukemia in primary and secondary recipients. Although in vivo no myeloid leukemias were observed, in vitro both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in chronic myeloid leukemia patients whereby a chronic myeloid phase can egress into a myeloid or lymphoid blast crisis. It is very plausible that differences between murine and human hematopoietic stem cell niches underlie these observed differences. Human engraftment in NSG mice is typically lymphoid biased, and since many growth factors and cytokines are species-specific it is clear that the murine niche is not ideal to evaluate human hematopoietic engraftment and leukemic transformation potential.In our current study we have evaluated the in vivo leukemic transformation potential of human CB derived CD34+ cells expressing BCR-ABL and/or BMI1 in NSG mice in which scaffolds coated with culture-expanded human mesenchymal stromal cells (MSCs) were implanted subcutaneously 8 weeks prior to injection of transduced cells, to allow the development of a humanized niche containing mineralized bone-matrix, osteoblasts, stromal cells, as well as appropriate vascularization (Groen et al., Blood 2012). BCR-ABL/BMI1 transduced human CB derived CD34+ cells or primary blast crisis CML patient cells were injected either intravenously or directly into the humanized scaffolds, and leukemia development was evaluated.Our data indicate that in a humanized niche, in contrast to a murine niche, BCR-ABL was sufficient to induce leukemia as a single hit without overexpression of exogenous BMI1. Furthermore, both ALL as well as erythro/myeloid leukemias could be induced. The ALL could be transplanted to secondary recipients and besides the lymphoid marker CD19, the cells also expressed CD33 and CD15, but not CD11b or GPA. These data are in sharp contrast to results obtained in xenograft mouse models without human niches, where BCR-ABL expression alone in human cells was not sufficient to induce leukemia, and secondary hits such as BMI1 were essential.Efficient engraftment of a blast-crisis CML patient sample was also observed in the human niche model, whereby the immature blast-like phenotype was maintained in the human scaffold niche, while more differentiated cells were observed in the mouse bone marrow niche. In vitro, long-term self-renewing cultures could readily be established with cells retrieved from the human scaffold niche of these leukemic mice, while no long-term cultures could be initiated with cells retrieved from the murine bone marrow niche, from the same mouse. These data indicate that a human niche is required to maintain appropriate in vivo self-renewal of human BC CML cells. Interestingly, the endogenous BMI1 levels were significantly higher in cells retrieved from the human scaffold niche as compared to the mouse BM niche.In conclusion, our data indicate that BCR-ABL transformed cells needs secondary event such as over expression of oncogene like BMI1 for its full transformation potential, most likely to overcome or repress oncogene-induced senescence. The mouse environment is not able to provide these secondary events in human cells, whereas the human niche is able to provide signals that together with BCR-ABL are sufficient to fully transform human cells in xenograft models. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Cell Lines and Patient Samples Show a Correlation Between Upregulated MSI2 and BCR-ABL Expression

Increased expression of Musashi 2 (also known as MSI2), a mRNA binding protein, is reported to trigger progression of chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC), which is frequently fatal. The data imply that MSI2 is upregulated by HOXA9 leading to disruption of the critical hematopoietic stem cell (HSC) fate decisions via the NUMB-NOTCH signaling pathway. These events lead to HSC proliferation, impaired myeloid differentiation and are associated with worse prognosis in CML and acute myeloid leukemia (Kharas, et al. Nat Med 2010; 16:903; Ito, et al. Nature 2010; 466:765). Previously, we confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in CP, irrespective of lymphoid or myeloid transformation (Kaeda, et al. Blood. 2011;118;supplement). To further elucidate this regulatory pathway we assessed whether HOXA9 expression correlated with increased MSI2mRNA levels and upregulation of NOTCH.Because of the rarity of BC samples and finding MSI2 increased in lymphoid and myeloid BC CML patients, we studied lymphoid [n=12 (Lymphoma:8; Myeloma:3; ALL:1)] and myeloid [n=12 (AML:4; CML:6; HES:1; ET:1)] cells lines. We also included 29 BCR-ABL positive (e1a2: 18; e13a2: 6; e14a2: 5) diagnostic samples from acute lymphoblastic leukaemia (ALL) patients (M:18; F:11); median age 50 years (range: 26-74). We quantified MSI2 and HOXA9 mRNA transcripts by quantitative PCR and expressed the data as % ratio of the control gene, GUSβ.Western blotting of cell line protein extracts was performed to assess expression of NOTCH 1, 2, 3 receptors and delta like ligand 3.We found MSI2 [median: 7.10% (range:0.06-38.71)] and HOXA9 [median: 2.40% (range:0.00-51.13)] expression was generally restricted to myeloid cell lines. Of the 12 lymphoid cell lines, MSI2 was only detectable in SUPB15 with 8.44%. The latter is a B-lymphocyte ALL cell line, known to express BCR-ABL e1a2 transcript. Similarly, HOXA9 [median: 0.00 (range:0.00-27.0%)] expression ranged from undetectable to 0.08%, among this group, apart from HD-MY-Z with 27.0%. In contrast, HOXA9 [median: 0.02% (range:0.01-2.18)] and MSI2 [median: 3.58% (range: 1.24-22.38)] transcripts were detectable in all 29 BCR-ABL positive ALL patients, irrespective of the transcript type expressed. Among the ALL samples 7 (24%) had increased MSI2 levels, i.e. >6.7% (Kaeda, et al. Blood. 2011;118;supplement) and of these 6 expressed e1a2 transcript and the other e13a2. In summary, upregulated MSI2 expression was observed in 17 (32.0%) of the 53 samples screened. But only 4 [KG1, EOL1, HEL and MEG-01 (all myeloid cell lines)] of the 17 also had increased HOXA9 levels. Remarkably, 5 (62%) of the 8 myeloid cell lines with increased MSI2 are known to express BCR-ABL. NOTCH1 receptor was detectable in all the lymphoid cell lines. But, NOTCH expression was highly variable in myeloid cell lines. Overall, an upregulated MSI2 mRNA expression was not reflected in the NOTCH receptor levels nor in the HOXA9levels.Our observations are consistent with MSI2 being limited to myeloid linage. However, in contrast to earlier reports our data imply that MSI2 functions via a pathway other than NOTCH signaling and is not regulated by HOXA9 alone. But the cell lines and ALL patients' data provide further evidence of correlation between MSI2 and BCR-ABL expression, suggesting they interact, directly or indirectly, to arrest cell differentiation and trigger BC. These findings, together with our reported data, show increased MSI2 levels to be an important biomarker of poor prognosis and are likely to have an impact in optimizing clinical management. It also represents a potential novel therapeutic target, especially for the BCR-ABL positive stem cells resistant to tyrosine kinase inhibitors. Disclosures:Kaeda:Novartis: Research Funding. le Coutre:Novartis: Research Funding.

Bilineal T lymphoblastic and myeloid blast transformation in chronic myeloid leukemia with TP53 mutation—an uncommon presentation in adults

Bilineal blast transformation of myeloid and T lymphoid type is a rare event in chronic myeloid leukemia. Here, we report a case in which an adult presented with high white cell counts and lymphadenopathy. Bone marrow studies confirmed the presence of 9 and 22 chromosomal translocation, and a diagnosis of chronic myeloid leukemia in chronic phase was made. Examination of a lymph node showed both myeloid and T lymphoblastoid blast crisis. Molecular studies demonstrated the presence of BCR-ABL fusion transcripts in both the myeloid and the T lymphoblastic component, indicating that the myeloid and T lymphoid blast crisis components shared common progenitors. TP53 deletion was demonstrated by fluorescence in situ hybridization.

Abstract IA20: Epigenetic deregulation of gene expression

Abstract Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ~20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss of function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCPG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using alpha-ketoglutarate (aKG) as a substrate. 2HG can disrupt the activity of enzymes that use aKG as a cofactor, including TET2 and the Jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibit TET2 hydroxymethylation and Jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards reduced histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is abundance in hematopoietic stem cells, and displays specific distribution patterns, and yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B-cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning. Citation Format: Ari Melnick. Epigenetic deregulation of gene expression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA20.

PDZ-RhoGEF and LARG Are Essential for Embryonic Development and Provide a Link between Thrombin and LPA Receptors and Rho Activation

G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.

Donor T Cells Administered Over HLA Class II Barriers Mediate Antitumor Immunity without Broad Off-Target Toxicity in a NOD/Scid Mouse Model of Acute Leukemia

Alloreactive (allo)-HLA–directed T cell responses after HLA-mismatched allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion are typically considered detrimental responses mediating graft-versus-host disease (GVHD). Allo-HLA-reactive T cells with beneficial and selective graft-versus-leukemia (GVL) reactivity, however, can also be identified within an HLA-mismatched context. We investigated whether allo-HLA class II–directed T cells with beneficial GVL reactivity induced in NOD/scid mice engrafted with human chronic myelogenous leukemia in lymphoid blast crisis after treatment with donor lymphocyte infusion – mediated detrimental xenogeneic GVHD as a result of broad off-target cross-reactivity. The results demonstrate that beneficial GVL reactivity and xenogeneic GVHD are mediated by separate T cells. GVL reactivity was mediated by human T cells recognizing allo-HLA class II molecules, whereas xenoreactivity was exerted by human T cells recognizing H-2 molecules. Taken together, our data indicate a limited risk for detrimental off-target effects by allo-HLA class II–directed T cells and thereby provide a basis for the development of strategies for selecting allo-HLA–restricted T cells with selective GVL reactivity for adoptive transfer after HLA-mismatched allogeneic hematopoietic stem cell transplantation.

Imatinib failure and response to dasatinib in a patient with chronic myeloid leukemia in blast crisis and a novel, nine-nucleotide BCR-ABL insertion mutation

Imatinib failure and response to dasatinib in a patient with chronic myeloid leukemia in blast crisis and a novel, nine-nucleotide BCR-ABL insertion mutation

Philadelphia positive chronic myeloid leukemia mimicking acute lymphoblastic leukemia in a child

Chronic myeloid leukemia (CML) is a rare hematological malignancy in children. The more common acute lymphoblastic leukemia (ALL) needs to be excluded in cases with lymphoid blast crisis phase of childhood CML. A 12-year-male child with generalized lymphadenopathy and hepatosplenomegaly had a crisis phase of presentation instead of the usual chronic phase of onset of disease. Hematological tests and molecular study were performed. Peripheral blood, bone marrow and lymph node fine-needle aspiration cytology smear showed features of CML in blast crisis. Blast cells were myeloperoxidase negative. Philadelphia chromosome with major BCR-ABL fusion product and B-cell lineage immunomarkers indicated CML origin of blasts rather than de novo ALL. Usually myeloid blast crisis is common in CML but the present case had lymphoid blast crisis which is less commonly documented. We report a case of ‘adult form of CML in the juvenile age group with lymphoid blast crisis and extramedullary blast proliferation.’

Pediatric chronic myeloid leukemia with B-cell lymphoid blast crisis at presentation

TO THE EDITOR: Chronic Myeloid Leukemia (CML) is a rare hematological malignancy accounting for less than 3% of pediatric and adolescent leukemias, with an annual incidence of approximately 1 per million children and young people aged 20% blasts in the marrow or the presence of extra-medullary blast proliferation [3, 4]. Blast transformation of CML is lymphoid in 30% of cases and myeloid in the remaining 70%. We report the case of a 10-year-old boy who presented with a 6-week history of weight loss and left-sided upper abdominal pain. On examination, he was found to have substantial splenomegaly extending into the right iliac fossa. His full blood count showed a hemoglobin level of 6.6 g/dL, a platelet count of 148×109/L, and a total white cell count of 575×109/L. A differential count showed the following: blasts, 30%; promyelocytes, 6%; myelocytes, 24%; metamyelocytes, 11%; neutrophils, 7%; eosinophils, 9%; basophils, 3%; monocytes, 1%; and lymphocytes, 10% (Fig. 1). Flow cytometry of the blast population showed that the blasts expressed CD19, cyCD79a, CD10, HLA-DR, CD34, and TdT surface antigens. Chromosome analysis showed a t(9;22)(q34;q11) translocation consistent with a Philadelphia chromosome (Fig. 2A). Interphase fluorescence in situ hybridization (FISH) showed BCR-ABL1 fusion signals in 90% of the nuclei (Fig. 2B). A minor breakpoint cluster region (210 kDa) was confirmed using RT-PCR. In the absence of a documented CML-CP, distinguishing between lymphoid blast crisis of CML and a Philadelphia chromosome-positive ALL can be difficult. A diagnosis of CML in B-cell lymphoid blast crisis rather than de novo precursor B-cell ALL was made because of the concurrent presence of basophilia, a predominance of metamyelocytes and myelocytes, and the p210 BCR-ABL transcript. The patient did not have any additional chromosomal anomalies associated with advanced phase CML and was negative for IgH rearrangement on FISH analysis. Fig. 1 Blood film demonstrating chronic myeloid leukemia in lymphoid blast transformation. Fig. 2 (A) Karyotype showing t(9;22)(q34;q11) Philadelphia translocation. (B) Interphase FISH, Vysis dual-fusion probe set. Green: BCR; red: ABL1; yellow: fused BCR and ABL signals corresponding to der(9) and der(22) translocation products. The patient achieved a complete hematological response and a minor cytogenetic response following induction therapy comprising dexamethasone, vincristine, daunorubicin, asparaginase, and imatinib. Nonetheless, he subsequently died of idiopathic pneumonitis after allogeneic stem-cell transplantation. The progression from CML-CP to CML-BC in the pre-tyrosine kinase era is well described, but CML-BC at presentation in children is extremely rare. The case reported here adds to the literature on the simultaneous presence of features of both lymphoblastic transformation and CML at presentation in the absence of cytogenetic clonal evolution. Further research into the biology of the aggressive phase of CML is required to develop novel targets to improve outcomes.

Can Activation Induced Cytidine Deaminase Lead to Genetic Instability in Bcr-Abl Negative Myeloproliferative Neoplasms?

AbstractAbstract 5056 Objective:Most patients with myeloproliferative neoplasms (MPN) have mutations that lead to constitutive activation of the tyrosine kinase signal transduction pathways. JAK2V617F mutation is present in about 97% of polycythemia vera (PV), and in 60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. JAK2-exon-12 mutations, MPL exon-10-mutations are also seen in MPNs. In addition to this mutations, some patients with MPN may acquire mutations of other genes, such as in genes involved in negative regulations of signaling pathways (LNK, CBL, SOCS1, SOCS2, SOCS3) and epigenetic regulations (TET2, ASXL1, DNMT3A, or EZH2). IKZF1 deletions and TP53 mutations are mainly found post-MPN acute myeloid leukemia (AML). Loss of function mutations in TET2, ASXL1 and EZH2 genes, may be early events preceding JAK2V617F but may also secondary genetic event. These mutations are more frequently observed in PMF than in PV and ET. They are also present in other myeloid malignancies such as myelodysplasia and AML. Moreover several cases have been described that are positive for BCR-ABL and JAK2V617F mutation. It is thought that the mutagenic phenotype existing inherently in patients with MPN can generate new mutations. With the contribution of these mutations, PV, ET and PMF might evolve into secondary myelofibrosis or leukemia at different ratios. Activation induced cytidine deaminase (AID) is required for a B cell terminal differentiation mechanism shared by somatic hypermutation and class-switch recombination. Aberrant expression of AID can induce imperfect mutations in non-immunoglobulin genes and was detected in human lymphomas, lung cancer and gastric or intestinal metaplasia. High AID expression is associated with poor prognosis in follicular lymphomas, chronic lymphocytic leukemia and promotes B lymphoid blast crisis in chronic myeloid leukemia. Current study aims to investigate AID expression levels in myeloproliferative neoplasms and their relationship to genetic instability in MPN patients. Material and methods:The patient group consisted of 120 patients with bcr-abl negative myeloproliferative neoplasms (PV: 32 patients, ET: 62 patients and PMF: 18 patients, post-ET or post-PV myelofibrosis: 5 patients, unclassified MPN: 1 patient, MPN-AML: 2 patients). The patients were admitted to Istanbul University, Istanbul Medical Faculty, Division of Hematology Outpatient Clinics. The control group consisted of 69 healthy persons. We evaluated AID expression levels of the patients and of the control group. RNA was extracted from peripheral blood samples of patients and controls, RNA was converted to complementary DNA (cDNA). Using the primers and probes of the target gene (AID) and reference gene HPRT (hypoxanthine phosphoribosyltransferase), cDNA was amplified by realtime polymerase chain reaction and expression levels of the genes were evaluated. Results:We found that AID expression levels in the patient group were significantly higher than in the control group (Target-AID/Reference-HPRT, mean value: 0. 1747 vs 0. 0417, respectively, p<0. 001). We found that the patients age, blood hemoglobin levels, history of thrombosis, bone marrow reticulin levels, serum LDH levels, organomegaly and having JAK2V617F mutation were not correlated with AID expression levels. Conclusion:We showed that AID expression levels in patient with myeloproliferative neoplasms were significantly higher than in healthy control group. The role and the biological significance of high AID expression levels in genetic instability in MPNs need to be elucidated. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Lineage-Specific Functions of LKB1 in CML and B Lymphoid Blast Crisis

Abstract 34 Background:The serine-threonine liver kinase B1 (LKB1, also called STK11) acts as negative regulator of aerobic glycoloysis, a metabolic pathway that is typically used in cancer cells (commonly referred to as ‘Warburg effect'). LKB1, together with AMPKs, shifts the metabolic balance from aerobic glycolysis to oxidative phosphorylation and thereby reverses the metabolic program of cancer cells and functions as tumor suppressor. Recently, it has been shown that LKB1 plays a critical role in the maintenance of quiescence and metabolic homeostasis of hematopoietic stem cells (HSCs). Hypothesis:In the present study, we focused on the roles of LKB1 in BCR-ABL1-driven leukemias including CML and B lymphoid blast crisis/Ph+ ALL (LBC). While LKB1 is widely seen as tumor suppressor in solid tumors, we found that high expression levels of LKB1 at diagnosis predict poor clinical outcome in patients with high risk acute lymphoblastic leukemia (n=207; COG P9906 trial; p=0.0204). In addition, high levels of LKB1 expression correlate with positive minimal residual disease (MRD+, p=0.0323) status in patients. These findings were unexpected and seem to contradict the common notion of LKB1 as a tumor suppressor. Results:To study the function of LKB1 in CML and B lymphoid blast crisis/Ph+ ALL (LBC), we developed a mouse model for inducible ablation of Lkb1 in BCR-ABL1-transformed hematopoietic stem and progenitor cells (CML-like) and B cell progenitors (LBC). To this end, Lkb1-fl/fl bone marrow hematopoietic stem and progenitor cells and B cell precursor cells were transformed with BCR-ABL1 and transduced with tamoxifen-inducible Cre. Unexpectedly, Cre-mediated deletion of Lkb1 had opposite effects in CML and LBC. While Lkb1-deletion in CML results in an initial proliferative burst of the leukemia cells, the vast majority of B cell lineage LBC cells undergo rapid cell cycle arrest. These findings are consistent with changes of cell cycle checkpoint proteins in response to Lkb1 deletion in CML and B cell lineage LBC. While Lkb1 deletion in CML cells results in downregulation of Arf, p53 and p27 levels, Lkb1 deletion in B lineage LBC cells resulted in upregulation of Arf and p27. In addition, Lkb1 deletion in CML resulted in inactivation of AMPK, a known substrate of LKB1, as well as enhanced activation of mTORC1. By contrast, while deletion of Lkb1 in B cell lineage LBC cells resulted in inactivation of AMPK as shown by reduced phosphorylation of AMPKα T172, there was reduction in mTORC1 activity based on diminished levels of phospho-p70 S6 kinase and S6 following LKB1 deletion.The effects of LKB1 on sensitivity of BCR-ABL1 CML and B lineage LBC cells to Imatinib were also examined. Lkb1-deficient ALL cells became more sensitive to Imatinib treatment. On the other hand, initial Lkb1 deletion rendered CML cells more resistant to Imatinib treatment. When primary patient-derived Ph+ ALL cells (n = 3) were treated with Imatinib, upregulation of phospho-LKB1 (S428) was observed. Finally, LKB1 was also shown to regulate energy homeostasis in CML and B cell lineage LBC in different manners, as measured by monitoring ATP and lactate production. Conclusions:Here we show that Lkb1 plays critical roles in mediating proliferation and cell growth in BCR-ABL1-driven leukemias. While LKB1 is widely seen as a tumor suppressor that limits aerobic glycolysis in cancer cells according to the Warburg effect, our findings demonstrate that LKB1 has lineage-specific functions in BCR-ABL1 driven leukemias. While LKB1 function in CML resembles its tumor suppressor function in solid tumors, LKB1 is critical for survival and proliferation on B cell lineage CML blast crisis and Ph+ ALL. The finding of a divergent role of Lkb1 in CML and B cell lineage LBC/Ph+ ALL is relevant because small molecule inhibitors of AMPK and mTORC1 are currently under development for the treatment of BCR-ABL1-driven leukemias. Disclosures:No relevant conflicts of interest to declare.

Long Term Follow-up of 121 Elderly Patients with Philadelphia-Positive Acute Lymphoblastic Leukaemia (Ph+ALL) Treated in Prospective GMALL Trials Supports a Greater Emphasis On Allogeneic SCT as Definitive Postremission Therapy.

AbstractAbstract 2608▪▪This icon denotes a clinically relevant abstract Background:Combined treatment with a tyrosine kinase inhibitor (TKI) and ALL-type induction and consolidation chemotherapy followed by allogeneic SCT is standard front-line therapy for younger patients with Ph+ALL, but the value of adding intensive chemotherapy to a TKI in elderly patients is controversial. More than 90% of elderly patients achieve a complete remission, irrespective of the type of TKI-based induction, but relapse is the major cause of treatment failure. In a previously reported randomized trial examining imatinib combined with intensive induction and consolidation chemotherapy for Ph+ALL in elderly patients (n=55), the probability of overall survival (OS) after 24 months was 42% ± 8% (Ottmann OG et al., Cancer. 2007; 109:2068-76). To date, very little published data on long-term outcome of elderly patients with Ph+ALL are available. Aims:We conducted the present analysis to determine whether subsets of patients derive long-term benefit from combined imatinib plus intensive chemoptherapy, examine the characteristics of long-term survivors, determine whether such patients can be identified by assessment of MRD, and obtain preliminary results on the feasibility and efficacy of SCT in this population of elderly patient. Study design and patients demographics:Our current analysis includes a total of 121 patients (119 ALL, 2 CML in lymphoid blast crisis), with a median age of 66 years (range 54–80). Fifty-five patients were enrolled in a previously reported randomized clinical trial comparing single-agent imatinib and chemotherapy as induction therapy, followed by up to 6 cycles of consolidation chemotherapy; a further 67 patients were subsequently treated according to this protocol as per recommendation by the GMALL Study Group. Results:The overall CR rate was 88%, median time to progression was 14.5 months (range 0.5–102) and OS was 18.6 months (range 0.5–102), respectively. Probabilities of remission duration, survival and TTP at 5 years were 19%, 22% and 19%, respectively. The type of initial induction therapy had no significant impact on OS and DFS. Of 113 pts, who were evaluable for comorbities, pulmonary disease was the only comorbidity associated with inferior outcome (median OS 13 months vs. 20 months, univariate analysis p=0.02). Allogeneic SCT was performed in CR1 in 12 patients and as salvage therapy in another 7 patients. Median age of these 19 patients was 62y (range 54–69). The time from diagnosis to SCT in CR1 was 4.6 months (2.9 mo – 16.8 mo) and from relapse to SCT in >CR1 3 months (2.1 mo – 6.1 mo).The 5yr OS in patients transplanted in CR1 vs. non-transplanted patients was superior (48% vs 22%). Remarkably, OS of the 7 patients transplanted beyond CR1 as part of salvage therapy was 43% after 4.5 years. With a median follow-up of 21.6 months (range 3.3– 54) after SCT, 8 patients are in ongoing CR with a median OS of 51.8 months from initial diagnosis (range 35 – 66), 5 pts. died in CR, 6 pts. relapsed. Conclusions:The combination of imatinib with intensive chemotherapy is feasible in elderly patients, but long-term survival is poor primarily due to high relapse rate. Allogeneic SCT in CR1 is superior to conventional therapy and should be considered as front-line therapy in this elderly patient population. The encouraging results of allogeneic SCT performed beyond CR1 suggest that SCT should be considered as definite postremission therapy in a larger proportion of elderly patients than is current practice. Disclosures:Ottmann:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

High Dose Cytarabine and Mitoxantrone in Combination with Dasatinib As Active Induction Therapy in Adult Patients with Philadelphia Chromosome Positive (ph+) Acute Lymphoblastic Leukemia (ALL)

AbstractAbstract 4293 Introduction:The Philadelphia chromosome is the most frequent cytogenetic abnormality in adult ALL, with an overall incidence of 20–30%. When treated with conventional therapy, adult patients with Ph+ ALL have an extremely poor prognosis. While current chemotherapy regimens typically induce complete responses (CRs) in the majority of patients, the quality of remission is poor as most patients relapse within 6–11 months of treatment and subsequently die from the disease. The 1 and 5-year overall survival rates for patients treated with imatinib-based chemotherapy regimens is approximately 60% and 30%, respectively.Previously we in collaboration with others have reported a Phase III randomized study of high-dose cytarabine and mitoxantrone (ALL-2 regimen) as induction therapy versus a more traditional treatment regimen with vincristine-prednisone based induction therapy in newly diagnosed adult ALL patients (Weiss, ASCO 2005). This study was conducted prior to the routine use of tyrosine kinase inhibitors (TKIs) for Ph+ disease. In that randomized study, there were 16 patients with Ph+ ALL treated with the frequency of CRs on the ALL-2 regimen 85% compared to 47% of patients treated on the vincristine-prednisone based arm. At 6 year follow-up, 26% of Ph+ patients treated with the ALL-2 regimen were alive compared to 0% of Ph+ ALL patients treated on the vincristine-prednisone induction arm.Based on this initial favorable response seen with the ALL-2 regimen in Ph+ patients and the emerging use of TKI therapy in Ph+ALL, dasatinib was added to this therapeutic regimen. This is the first report of this combination in newly diagnosed patients with Ph+ ALL or blastic phase CML patients. Methods:Eligible patients included previously untreated or treated adults with a diagnosis of Ph+ ALL or lymphoid blast crisis of known CML. Patients must have evidence of t(9;22) in leukemic cells based on chromosomal or molecular analysis. Patients received induction therapy with high-dose cytarabine (3000 mg/m2 IVPB daily, days 1–5) and mitoxantrone (80mg/m2 IVPB on day 3 for patients ≤ 65 yrs or 40mg/m2 for patients >65 yrs) in combination with dasatinib. Patients in the phase I portion of the study were assigned to one of 3 successive dose cohorts of dasatinib: dose level 1: 70mg daily, dose level 2: 100mg daily; dose level 3: 140mg daily, starting on day 1. Patients then received treatment with Consolidation A: vincristine, dexamethasone, and dasatinib. Following consolidation A, patients who achieved a CR and were ≤ 70 years of age and had a suitable HLA donor were referred for allogeneic stem cell transplant. The remaining patients continued consolidation and maintenance therapy per the ALL-2 regimen with dasatinib as per protocol. Results:Seven patients with Ph+ disease were enrolled between 3/2010-11/2011 (Table 1). All (100%) achieved complete remission after induction; 6 of the seven patients (86%) were in both cytogenetic and molecular remission. Although the numbers are small, there does not seem to be a difference in response depending upon the dose level of dasatinib administered. One patient was taken off study due to recurrent gastrointestinal issues but continued to be followed for response and survival and completed induction therapy as per protocol. Six of the seven patients (86%) went on to receive allogeneic stem cell transplant in first remission. The patient who was ineligible for allogeneic stem cell transplant due to multiple comorbidities continues on maintenance chemotherapy as per protocol. Two patients have died in remission due to post transplant complications. One patient became MRD positive post-transplant and is on nilotinib therapy. Median overall survival has not been reached with median follow-up greater than 20 months. Conclusions:The addition of dasatinib to high-dose cytarabine and mitoxantrone induction therapy for patients with Ph+ ALL is tolerable and efficacious. Patients achieved a higher frequency of CRs compared to our historical study that did not use TKI-based therapy in this patient population. Long-term follow-up of these patients is ongoing.Patient CharacteristicsAge (median)39–64 (median 57)Sex3 male; 4 femaleCytogenetics:Ph+ ALL p1906Blastic CML p2101Other abnormalities notedMonosomy 7 in 2 patients; abnl der 9Frequency of CR100%Frequency of molecular remission71%Frequency of cytogenetic remission100%Received BMT in first CR6Overall Survival71% Disclosures:Lamanna:Bristol Meyers Squibb: Research Funding. Off Label Use: dasatinib as part of initial therapy for patients with Philadelphia chromosome + ALL.

The Etiology of Pleural Effusions Develping During Dasatinib Therapy in CML Could Be Variable: Need for Awareness and Investigations

Abstract 4456Dasatinib is a highly effective oral TKI for CML at various stages of the disease. The most common non-hematologic side-effect is pleural effusion. The exact mechanism of this pathology remains to be fully elicited, although studies report it to be an immunological phenomenon. Current literature shows dasatinib alone to be the cause of such effusions. More recently, pulmonary hypertensions have been reported on dasatinib therapy.However, in our cohort of CML patients receiving dasatinib as a first or second line therapy, other pathologies were documented. Grade 2/3 unilateral pleural effusions were detected in 3 patients. Case 1.A 31 yr old male with CML-CP was on first line dasatinib 100 mg OD and CCyR was documented at 3 months. At 6 month, he was detected to have grade 1/2 bilateral pleural effusion and was managed with drug interruption and oral dexamethasone with no significant reduction of fluid. CBC was normal. Dasatinib was continued. At one year, he lost cytognetic response with emergence of complex karyotype and medullary lymphoid blast crisis was confirmed. Pleural fluid showed lymphoblasts proven by cytology and immunophenotype. He received vincristine and prednisone followed by a change to imatinib. His disease progressed further and died at 2 years from diagnosis. Case 2.A 39 yr old male with molecular relapse and M244V Abl Kd mutation at 8 years since diagnosis and 6.5 yrs on imatinib 400–800 mg daily was offered dasatinib 100 mg OD. After 24 months, he developed grade2 left pleural effusion which responded poorly to drug interruption, therapeutic tapping and prednisone. A few months later he developed constitutional symptoms and CT chest, bronchoscopy/BAL proved to have left lung tuberculosis. Currently, he is responding favorably to anti-tubercular therapy. He was switched back to imatinib and remains in cytogenetic remission. Case 3.A 21 yr old male with cytogenetic non-responder to imatinib 800 mg daily was put on dasatinib 100 mg OD. An MMR was achieved. He developed left pleural effusion after 4 years of dasatinib. Extensive investigation for any specific pathology was not productive. He was put on empiric anti-tubercular therapy with no response. It is being treated as a dasatinib induced effusion. Conclusions.During dasatinib therapy, the causes of pleural effusions could be varied – drug related, extramedullary blast crisis or tubercular. Radiological, cytological and microbiological investigations could be indicated after careful clinical evaluation. Disclosures:Saikia:Bristol -Myers Squibb: Research Funding, Speakers Bureau.

Dasatinib combined with weekly administration of vincristine as effective therapy in sudden or resistant Ph+ lymphoid blast crisis of chronic myeloid leukaemia

Dasatinib combined with weekly administration of vincristine as effective therapy in sudden or resistant Ph+ lymphoid blast crisis of chronic myeloid leukaemia

Transcriptional suppression of BACH2 by the Bcr-Abl oncoprotein is mediated by PAX5

Bach2 is a lymphoid-specific transcription factor with a prominent role in B-cell development and apoptosis-induction in response to oxidative stress. We previously showed that Bach2 is downregulated in chronic myeloid leukaemia (CML), and here we demonstrate the mechanism by which Bcr-Abl mediates this phenomenon. We have cloned a 3.9 Kb genomic DNA fragment upstream of the transcription initiation site, and delineated the core and proximal BACH2 promoter regions. Transient BCR-ABL expression led to significant reduction in BACH2 promoter activity and this effect was dependent on the kinase function of the oncoprotein. Sequential deletions disclosed several regulatory elements within the promoter region, as well as within BACH2 exonic sequences. Analysis of these elements and transient transfection assays led to the identification of the Pax5 transcription factor as a potent trans-activator of BACH2, whose effect is predominantly mediated through occupation of a binding site on the BACH2 promoter, as demonstrated by both in vitro and in vivo experiments. Overall, our data show that Pax5 functions as an intermediate effector in the Bcr-Abl-mediated transcriptional repression of BACH2. The current results, combined with previous reports, establish Pax5 and Bach2 as transcriptional targets of Bcr-Abl, whose downregulation may contribute to lymphoid blast crisis of CML.

Abstract 3839: Nuclear export (karyopherin) inhibitors: A novel therapeutic strategy for treating Philadelphia-positive (Ph+) acute leukemias

Abstract Tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis (BC) chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Increased expression/activity of nucleocytoplasmic-shuttling heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, are critical for the expression of factors (e.g. SET/PP2A, C/EBPβ/miR-328, and c-Myc) regulating proliferation and survival of Ph+ progenitors. Because the karyopherin CRM1 controls nuclear export of hnRNPs, we assessed the therapeutic potential of CRM1 inhibitors in CML-BC and Ph+ ALL models. Thus, myeloid 32D-p210BCR-ABL1 and lymphoid Baf3-p190BCR-ABL1 progenitors were exposed to the CRM1 selective &amp; potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays revealed that KPT-185 and KPT-207 decreased cell viability by ∼80% at concentrations ranging from 150-350 nM. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70-100% viable cells) by KPT-SINE. As expected, treatment (1 μM; 48h) with these inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. As a result, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors. Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization, which is important for regulation of the PP2A inhibitor SET and, therefore, for BCR-ABL1 leukemogenesis, we assessed whether KPT-207 and KPT-185 negatively regulate PP2A activity. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells. Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3839. doi:1538-7445.AM2012-3839