Chronic Myeloid Leukemia Blast Crisis Research Articles

Abstract 3338: Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche

Abstract Introduction Pacritinib, a potent clinical small molecule inhibitor of JAK2, it also suppresses signaling through wild-type and mutant FLT3, IRAK1, and CSF1R. Pacritinib does not cause marrow suppression and has demonstrated single agent activity preclinically in myelofibrosis and other myeloid neoplasms including AML and CMML. Stromal protection was not observed. However, the capacity of pacritinib to eradicate therapy resistant leukemia stem cells (LSC), residing in the bone marrow niche, had not been examined. Thus, we investigated the impact of pacritinib alone or in combination with standard of care therapy on primary blast crisis chronic myeloid leukemia (BC CML), myelofibrosis (MF) and AML LSC survival and self-renewal in a stem cell supportive niche. Methods Genetically engineered mouse bone marrow fibroblasts producing human SCF, IL3 and G-CSF were used as stromal monolayers to support LSC survival and self-renewal. Human primary CD34+ cells were selected from BC CML (n = 5), MF (n = 5) and relapsed AML (n = 4) before and after clinical treatment with azacitidine. As a control, CD34+ cells from age matched normal bone marrow (a-NBM, n = 4) were used for the co-culture. Survival and self-renewal of the cells were investigated by colony forming and replating assays. Pacritinib was used at concentrations ranging from 10 to 50 nM alone and in combination with 1 nM dasatinib. Results Pacritinib alone induced dose-dependent inhibition of self-renewal in a-NBM, AML, MF and CML-BC, with the optimal concentration of 20nM leading to IC50 diversity in the response between normal and leukemia progenitors. AML and MF responded uniformly and inhibition reached 50% at 10nM concentration. BC CML cells were more divergent: 40% demonstrated >50% inhibition, in another 40% it was 20-50% and in 20% inhibition was <20%. Combined treatment with the low dose of dasatinib and pacritinib doses of 10 or 20 nM resulted in a significant (p<0.001, Anova) difference in self-renewal of all BC CML cells, raising a possibility of an additive/synergistic mechanisms. AML cells collected before and after clinical treatment with azacitidine uniformly showed a significant decrease in self-renewal starting with 10 nM pacritinib alone and combined treatment with dasatinib did not enhance the inhibition, suggesting a prospect of using pacritinib as a single agent in the treatment of relapsed AML. Conclusions Together these data indicate that possibly through inhibition of CSF1 and IRAK1 signaling in addition to suppression of JAK2, even in the presence of a LSC supportive niche, readily clinically achievable low nM concentrations of pacritinib alone are effective in reducing self-renewal of MF and relapsed AML. However, a combination of dasatinib and pacritinib is required to eliminate self-renewing LSC in BC CML with minimal toxicity toward normal progenitors. Targeting niche-dependent signaling could represent a robust avenue for treatment of refractory myeloid leukemia. Citation Format: Larisa Balaian, Anna Kulijian, Edward D. Ball, Catriona H.M Jamieson. Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3338.

Abstract 1931: MiR182 mediated control over myeloid differentiation provides novel mechanism of Imatinib resistance in chronic myeloid leukemia

Abstract MiR182 is an evolutionarily conserved miR, present in a cluster with miR183, and miR96 on human chromosome 7q32.2. This cluster is over-expressed in hESCs, and iPSCs. Developmentally, miR182 is over-expressed during erythropoiesis from CD34 cells. Erythroid differentiation of CML cells induced by Imatinib treatment provides an alternate mechanism of Imatinib escape, and also emphasizes the need to explore miR182 function in this context. In cancerous cells, miR182 regulates metastasis in melanoma cells by regulation of MITF and FOXO3, and DNA repair in breast cancer by targeting BRCA1. These reports provide some understanding of miR182 function by transient in-vitro assays. To refine functional importance, targeted deletion of MIR182 has been done on mice retinal cells where it is shown an over-expressed candidate by quantitative methods; nevertheless MIR182 deletion does not show any phenotypic change indicating challenges to study their functions along with better model systems. We focus to study K562 cells, CML blast crisis cell line with wild type Bcr-Abl tyrosine kinase, partially differentiated cells able to differentiate in different lineages upon induction. We exploit use of CRISPR/Cas9 mediated knock-out approach to find out miR182 function in K562 cells. We show that we have successfully deleted MIR182 loci in K562 cells by CRISPR. We hypothesize if miR182 regulates proliferation or differentiation in bi-potent K562 cells. Using phenotypic characterization, we studied MIR182 deleted cells. We show that myeloid differentiation is augmented by MIR182 deletion. Homozygous MIR182 deleted cells display decreased proliferation. We find striking inverse correlation with notch signalling genes in the context of Imatinib resistance. Notch signalling has been documented in cancer cells where its role has been shown in context dependent which require more explanation of its regulation. Hes1, one of main transcriptional regulator of notch signalling pathway, is targeted by miR182 shown by both bio-informatics, and in-vitro assays. Hes1 manipulation confirms it as down-stream target of miR182 in CML cells. We next explored Imatinib resistance phenotype in the context of miR182 given its expression in CD34 cells. We demonstrate miR182 is over-expressed in CML cells towards Imatinib treatment in ex-vivo, and in-vitro. We find miR182 essential for proliferation of K562 cells, Imatinib resistance. Taken together, our studies highlight miR182 targets notch signalling genes in CML cells implication of which we have shown in the context of Bcr-Abl independent Imatinib resistance mediated by differentiation deregulation. Citation Format: Deepak Arya, P. Sasikala, Shang Li, Dasaradhi Palakodeti, Cecil Ross, Sudhir Krishna. MiR182 mediated control over myeloid differentiation provides novel mechanism of Imatinib resistance in chronic myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1931.

Transplantation from haploidentical donor is not inferior to that from identical sibling donor for patients with chronic myeloid leukemia in blast crisis or chronic phase from blast crisis.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for chronic myeloid leukemia (CML) patients in blast crisis (BC), and haploidentical donors (HID) are immediately available for most patients. We compared the outcomes of HID transplantation with those of matched related donor (MRD) transplantation in a cohort study. A total of 90 consecutive patients who received allogeneic HSCT because of CML-BC were investigated retrospectively. A total of 67 patients underwent transplantation from HID and 23 from MRD. Survival outcomes were compared between the two cohorts. Of the 90 patients, 86 patients were engrafted. Three-year overall survival (OS) and relapse-free survival (RFS) were comparable between HID and MRD recipients (OS: 60.0% vs 55.3%, respectively, P=.580; RFS: 51.1% vs 47.8%, respectively, P=.512). Three-year incidences of transplant-related mortality (TRM) and relapse did not differ between HID and MRD recipients (relapse: 21.0% vs 26.1%, respectively, P=.626; TRM: 27.9% vs 26.1%, respectively, P=.937). In multivariate analyses, previous chemotherapy history and not achieving CHR before HSCT are independent adverse predictors of OS. For CML-blast crisis or chronic phase from blast crisis patients, HID transplantation achieves comparable survival to MRD transplantation. HID donors can be regarded as regular donors for these special patients at selected centers.

Leukemic Stem Cells Evade Chemotherapy by Metabolic Adaptation to an Adipose Tissue Niche

Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36(+) LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

BCR-ABL-positive acute myeloid leukemia: a new entity? Analysis of clinical and molecular features.

BCR-ABL-positive acute myeloid leukemia (AML) is a rare subtype of AML that is now included as a provisional entity in the 2016 revised WHO classification of myeloid malignancies. Since a clear distinction between de novo BCR-ABL+ AML and chronic myeloid leukemia (CML) blast crisis is challenging in many cases, the existence of de novo BCR-ABL+ AML has been a matter of debate for a long time. However, there is increasing evidence suggesting that BCR-ABL+ AML is in fact a distinct subgroup of AML. In this study, we analyzed all published cases since 1975 as well as cases from our institution in order to present common clinical and molecular features of this rare disease. Our analysis shows that BCR-ABL predominantly occurs in AML-NOS, CBF leukemia, and AML with myelodysplasia-related changes. The most common BCR-ABL transcripts (p190 and p210) are nearly equally distributed. Based on the analysis of published data, we provide a clinical algorithm for the initial differential diagnosis of BCR-ABL+ AML. The prognosis of BCR-ABL+ AML seems to depend on the cytogenetic and/or molecular background rather than on BCR-ABL itself. A therapy with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, or nilotinib is reasonable, but-due to a lack of systematic clinical data-their use cannot be routinely recommended in first-line therapy. Beyond first-line treatment of AML, the use of TKI remains an individual decision, both in combination with intensive chemotherapy and/or as a bridge to allogeneic stem cell transplantation. In each single case, potential benefits have to be weighed against potential risks.

ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1's effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.

High IL-7 levels in the bone marrow microenvironment mediate imatinib resistance and predict disease progression in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a three-stage myeloproliferative disease caused by translocation between chromosomes 9 and 22. Although tyrosine kinase inhibitors (TKI) are highly effective in the treatment of CML, numerous clinical trials have shown that many patients become refractory or drug resistance, especially those in the blastic crisis of CML. The molecular mechanisms underlying CML, however, remain poorly understood. In the present study, we used a coculture model to address possible mechanisms underlying the involvement of bone marrow microenvironment in the drug resistance of CML. Our data show that interleukin-7(IL-7) levels in the bone marrow of CML patients in blastic crisis are significantly higher than those of both healthy persons and CML patients in chronic and accelerated phases. The increased IL-7 was secreted by mesenchymal stem cells (MSC) in the bone marrow, which may protect leukemic cells from apoptosis induced by imatinib through JAK1/STAT5 signaling pathway. Our findings suggest that therapeutic strategies IL-7 signaling pathway may represent a promising approach for improving CML therapy, especially for patients in blastic crisis.

Modeling BCR-ABL and MLL-AF9 leukemia in a human bone marrow-like scaffold-based xenograft model.

Although NOD-SCID IL2Rγ-/- (NSG) xenograft mice are currently the most frequently used model to study human leukemia in vivo, the absence of a human niche severely hampers faithful recapitulation of the disease. We used NSG mice in which ceramic scaffolds seeded with human mesenchymal stromal cells were implanted to generate a human bone marrow (huBM-sc)-like niche. We observed that, in contrast to the murine bone marrow (mBM) niche, the expression of BCR-ABL or MLL-AF9 was sufficient to induce both primary acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Stemness was preserved within the human niches as demonstrated by serial transplantation assays. Efficient engraftment of AML MLL-AF9 and blast-crisis chronic myeloid leukemia patient cells was also observed, whereby the immature blast-like phenotype was maintained in the huBM-sc niche but to a much lesser extent in mBM niches. We compared transcriptomes of leukemias derived from mBM niches versus leukemias from huBM-like scaffold-based niches, which revealed striking differences in the expression of genes associated with hypoxia, mitochondria and metabolism. Finally, we utilized the huBM-sc MLL-AF9 B-ALL model to evaluate the efficacy of the I-BET151 inhibitor in vivo. In conclusion, we have established human niche models in which the myeloid and lymphoid features of BCR-ABL+ and MLL-AF9+ leukemias can be studied in detail.

Aberrant activation of CaMKIIγ accelerates chronic myeloid leukemia blast crisis.

Blast crisis (BC) is the final deadly phase of chronic myeloid leukemia (CML), but its molecular basis remains poorly understood. Here, we show that CML BC is regulated by calcium-calmodulin-dependent kinase IIγ (CaMKIIγ). Genetic deletion of CaMKIIγ greatly inhibits disease progression via selectively impairing the self-renewal of leukemia stem cells (LSCs) in mouse models, whereas overexpression of CaMKIIγ has the opposite effects. In human CML, phosphorylated CaMKIIγ abundance is significantly associated with BC. Moreover, CaMKIIγ phosphorylates and reduces the nuclear cyclin-dependent kinase inhibitor p27Kip1, a critical brake that maintains LSC quiescence. These findings suggest that CaMKIIγ might be an important switch for the transition of CML BC and identify a unique mechanism by which CaMKIIγ promotes the self-renewal of LSCs by deceasing nuclear p27Kip1 to wake up dormant LSCs. Therefore, CaMKIIγ may provide a new therapeutic target to treat CML BC.

NUMB inactivation confers resistance to imatinib in chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase, where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and its derivatives. NUMB is an evolutionary well-conserved protein initially described as a functional antagonist of NOTCH function. NUMB is an endocytic protein associated with receptor internalization, involved in multiple cellular functions. It has been reported that MSI2 protein, a NUMB inhibitor, is upregulated in CML blast crisis, whereas NUMB itself is downregulated. This suggest that NUMB plays a role in the malignant progression of CML. Here we have generated K562 cells (derived from CML in blast crisis) constitutively expressing a dominant negative form of NUMB (dnNUMB). We show that dnNUMB expression confers a high proliferative phenotype to the cells. Importantly, dnNUMB triggers a partial resistance to imatinib in these cells, antagonizing the apoptosis mediated by the drug. Interestingly, imatinib resistance is not linked to p53 status or NOTCH signaling, as K562 lack p53 and imatinib resistance is reproduced in the presence of NOTCH inhibitors. Taken together, our data support the hypothesis that NUMB activation could be a new therapeutic target in CML.

New Developments in Chronic Myeloid Leukemia: Implications for Therapy.

Context:Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by overproduction of immature and matured myeloid cells in the peripheral blood, bone marrow and spleen.Evidence Acquisition:A hallmark of CML is the presence of (9; 22) (q34; q11) reciprocal translocation, which is cytogenetically visible as Philadelphia chromosome (Ph) and results in the formation of BCR-ABL1 fusion protein. This fusion protein is a constitutively active tyrosine kinase which is necessary and sufficient for malignant transformation. The introduction of imatinib, a BCR-ABL1- targeting tyrosine kinase inhibitor (TKI) has revolutionized CML therapy. Subsequently, two other TKIs with increased activity against BCR-ABL1, dasatinib and nilotinib, were developed and approved for CML patients. Nevertheless, CML therapy faces major challenges.Results:The first is the development of resistance to BCR-ABL1 inhibitors in some patients, which can be due to BCR-ABL1 overexpression, differences in cellular drug influx and efflux, activation of alternative signaling pathways, or emergence of BCR-ABL1 kinase domain mutations during TKI treatment. The second is the limited efficiency of BCR-ABL1-TKIs in blast crisis (BC) CML. The third is the insensitivity of CML stem cells to BCR-ABL1 inhibitors. Conventional chemotherapeutics and BCR-ABL1 inhibitors which act by inhibiting cell proliferation and inducing apoptosis, are ineffective against quiescent CML stem cells.Conclusions:A better understanding of the mechanisms that underlie TKI resistance, progression to BC, genomic instability and stem cell quiescence is essential to develop curative strategies for patients with CML.

Shoulder Myeloid Sarcoma: An Initial Presentation of CML Blast Crisis.

Myeloid sarcoma (MS) represents extra medullary accumulation of the immature cells of granulocytic series and occurs most commonly in the setting of acute myelogenous leukemia. Its occurrence in chronic myeloid leukemia (CML), myelodysplastic syndrome and other myeloproliferative neoplasm is uncommon. We here in report a 35-year old lady who was diagnosed as CML-chronic phase (CP) in the year 2004 and was on imatinib (400mg OD) since then with regular follow up and good compliance. She had progression to accelerated phase in April 2014 which was managed by increasing the dose of imatinib to 600mg OD. In August 2015, she presented with complaints of pain and swelling of the left shoulder suggestive of septic arthritis. Investigations revealed an ill-defined lesion involving muscles around the shoulder and clavicle. Absence of response to antibiotics and negative work up for infectious etiology raised the suspicion for MS which was later confirmed by immunohistochemistry of the aspirate from the lesion. Bone marrow examination was consistent with CML-CP. Hence, the diagnosis of CML with extra medullary blast crises was made. Patient was treated with a combination of high dose imatinib, hydroxyurea, cytarabine and local radiotherapy. Rarity of MS involving the shoulder and it's resemblance to septic arthritis has been highlighted in the present case. We emphasize the importance of immunohistochemistry of the aspirate for the timely and correct diagnosis of the cases who do not respond to an initial antibiotic trial.

Characteristics and clinical outcome of T315I mutation in Philadelphia chromosome-positive acute lymphoblastic leukemia and chronic myeloid leukemia

目的分析伴T315I突变的Ph染色体阳性急性淋巴细胞白血病(Ph+ALL)和慢性髓性白血病(CML)的特征及疗效。方法收集2014年3月至2015年6月于郑州大学附属肿瘤医院行ABL激酶区突变检测并对酪氨酸激酶抑制剂(TKI)耐药的23例Ph+ ALL患者和95例CML患者的临床资料。采用R显带法进行染色体分析，实时定量PCR方法检测BCR-ABL融合基因，TRIzol法提取总RNA，直接测序法检测ABL激酶区突变。结果ABL激酶区突变发生率在TKI耐药Ph+ ALL和CML中分别为60.9%(14/23)、41.1%(39/95)，其中T315I突变发生率分别为34.8%(8/23)和5.3%(5/95)，差异具有统计学意义(χ2=13.586，P<0.01)。CML慢性期患者ABL激酶区突变发生率为38.8%(19/49)，加速期、急变期分别为47.1%(8/17)、41.4%(12/29)，差异无统计学意义(χ2=0.360，P=0.835)。Ph+ ALL、CML患者自开始TKI治疗至发生T315I突变的中位时间分别为10和19个月，T315I突变发生至死亡或随访终止的中位时间分别为2和3个月，中位血液学缓解持续时间分别为10和16个月，中位总生存时间分别为13和42个月。结论Ph+ ALL较CML更易出现T315I突变，但两者自开始TKI治疗至发生T315I突变的中位时间相近，在现有方案治疗下，两者血液学缓解持续时间、总生存时间相近。

Immunophenotyping in Chronic Myeloid Leukemia Blast Crisis: Looking beyond Morphology

ABSTRACT Introduction Patients of chronic myeloid leukemia (CML) eventually transform to blast crisis (BC) after a variable length of chronic phase (CP). With improvement in management protocols, many patients of CML-BC are expected to show remission after initial chemotherapy and a better survival. Hence, it becomes imperative to correctly classify the nature of BC for further course of management. Materials and methods This study retrospectively analyzed cases of CML-BC, over a period of 5 years, with comprehensive immunophenotyping done on flow cytometry. Results Fifteen cases of CML-BC with male-to-female ratio of 3:1 and mean age of 40.2 years were included in the study. Flow cytometry revealed 14 cases of myeloid and one case of lymphoid BC. One case each of megakaryocytic and myelomonocytic lineage were seen among the 14 cases of myeloid BC. Aberrant marker expression was a common occurrence. Myeloperoxidase cytochemical stain had shown positivity only in 3 of the 14 cases of myeloid BC. Conclusion Immunophenotyping is necessary for assigning specific lineage to blasts in patients of CML-BC and thus providing clinically useful information regarding the treatment protocols and prognosis of the patient. How to cite this article Narang V, Sachdeva MUS, Bose P, Varma N, Malhotra P, Varma S. Immunophenotyping in Chronic Myeloid Leukemia Blast Crisis: Looking beyond Morphology. J Postgrad Med Edu Res 2016;50(4):181-184

A Convenient Cell Culture Model for CML Acquired Resistance Through BCR-ABL Mutations.

Tyrosine kinase inhibitors (TKIs) are the effective treatments for chronic myeloid leukemia (CML). However, clinical resistance to TKIs that leads to patient relapse remains a challenge. Acquisition of BCR-ABL mutations is crucial in the resistance but the underlying molecular mechanisms are poorly understood. Here we describe a cell culture model for CML acquired resistance in which blast crisis CML cells undergo initial apoptosis upon treatment with therapeutically effective doses of TKIs, but the cells regrow quickly with development of resistance through BCR-ABL mutations. This model mimics the clinical process of acquisition of BCR-ABL mutations and will be an important tool to dissect molecular mechanisms of CML drug resistance and to explore strategies to overcome resistance.

Isolated CNS Blast Crisis of CML in A Patient on Dasatinib Therapy

We report a case of a young male with chronic phase CML who despite achieving an excellent response to dasatinib therapy, developed isolated CNS blast crisis even though this tyrosine kinase inhibitor is the only one reported to cross the blood brain barrier.

B-cell Acute Lymphoblastic Leukemia With t(9;22)(q34;q11) Translocation and Clonal Divergence Through ider(22) Chromosome and t(13;17)(q14;q25) Translocation

Dear Editor, The Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (MDBGFC) has more than 100 cases for the t(9;22)(q34;q11.2) with divergent patterns of clonal evolution [1]. Here, we present a patient with B-cell ALL who showed t(9;22)(q34;q11.2) and a clonal divergence. Hematological studies from a 54-yr-old woman revealed white blood cell count of 97.2×109/L, platelets 35×109/L, and hemoglobin 6.9 g/dL. Bone marrow blasts displayed L2 morphology and expressed cell-surface differentiation antigens defining a B-cell phenotype, namely, CD10 (80%), CD19 (78%), CD5 (11%), CD3 (0%), and CD13 (0%). The patient received vincristine, prednisone, and daunorubicin-based chemotherapy. However, four months later, the patient was in the terminal phase and died after infiltrations were detected both to the retro-ocular and central nervous systems. We analyzed metaphasic cells from non-stimulated peripheral blood samples obtained eight days before the patient's death. Her karyotype included several sub-clones, which were described according to the International System for Human Cytogenetic Nomenclature (ISCN) (2013) [2], as: 46,XX,t(9;22)(q34;q11.2)[3]/46,idem,ider(22)(22pter→22q11.2::9q34→9q?tel::9q?tel→9q34::22q11.2→22pter)[14]/46,idem,t(13;17)(q14;q25)[3]/46,idem,+1,dic(1;1)(?;?),t(13;17)(q14;q25)[8]/46,XX[1] (Fig. 1A-E). C-banding by barium hydroxide and Giemsa revealed two heterochromatic regions (1q12 band) on the dic(1;1) chromosome (Fig. 1F). Fig. 1 Chromosomes banded with standard techniques. (A) Metaphasic cell with the ider(22). (B) This metaphasic cell contains both t(9;22)(q34;q11.2) and t(13;17)(q14;q25). (C) In addition to the two translocations observed in the above image, a dic(1;1) chromosome ... FISH studies with the dual-color, single-fusion BCR/ABL1 probe (Vysis, Downers Grove, IL, USA) revealed two fusion signals on the ider(22) and only one fusion signal on the standard Philadelphia chromosome (data not shown). FISH study with the RB1 probe (Vysis) disclosed two signals on both normal 13 chromosomes in the clone containing ider(22) (Fig. 2A). Cells with t(13;17) were positive only on the normal chromosome 13 and did not display any signal on either der(13) or der(17) (Fig. 2B and C), thus demonstrating the loss of RB1 by translocation. Fig. 2 FISH analysis with the RB1 probe (red signals) in cells counterstained with DAPI (4',6-diamidino-2-phenylindole; blue color). (A) Metaphase from the clone with ider(22) showing two RB1 signals on both normal 13 chromosomes. (B) Cell with t(13;17) displaying ... Yamamoto et al. [3] reported the presence of first and only ider(22) chromosome similar to the one observed by us in another ALL patient who had a poor prognosis too. These ider(22) chromosomes could result from an U-type sister chromatid exchange. The ider(22) chromosome equals a second copy of der(22) chromosome, which is well known to be one of the main secondary chromosomal changes related to the clonal evolution of cells with t(9;22) [1]. The co-occurrence of t(9;22)(q34;q11) and t(13q14) is rare. There are only six cases registered in the MDBGFC [1], which were previously reported [4,5,6,7]. When both translocations were present, the patients had CML in blast crisis or relapse. Out of four patients tested for chromosome 13 deletions by using FISH [5,6], three tested negative, and only in the fourth patient, there was inconclusive evidence of such a deletion [6]. The t(13;17)(q14;q25), which caused the loss of RB1 sequences in the present patient has not been previously described in ALL [1]. This translocation has only been detected in an AML-M4 patient who did not show deletion of the RB1 gene [8], and in a case of Sezary syndrome in which the status of RB1 gene was not tested [9]. Although we did not know the extent of present 13q deletion, some features of the patient such as genome instability, poor prognosis, and shortened survival suggest that she had a large deletion inclusive of the RB1 gene. Moreover, the chromosomal band 17q25 has been implicated in several translocations also associated with poor prognosis and shorter survival [1]. Imbalances in chromosome 1 are frequent in human neoplasia [1]. The apparently different sizes of two heterochromatic regions observed on the dic (1;1) chromosome (Fig. 1F and ​and2C)2C) suggest a translocation between homologous chromosomes as the forming mechanism of such a dicentric chromosome. Concomitant with the clonal divergence found in peripheral blood, this patient had central nervous system and retro-ocular infiltrations that could not be further studied. Nowicki et al. [10] observed that the circulating CML-blast crisis cells showed major changes than the CML-cells from bone marrow. This observation suggests that leukemic bloodstream cells differ from those in their original niche in survival, growth, invasiveness, and metastatic potential. In our patient, we could not determine whether the divergent sub-clones found in peripheral blood were also present in the infiltrations, or whether each infiltration was derived from a unique and independent sub-clone. This case calls for research on chromosomal and genetic changes that enable the leukemic cells to infiltrate other tissues.

Molecular Characterization of Philadelphia Chromosome Positive Acute Myeloid Leukemia - New Provisional Entity?

IntroductionPhiladelphia chromosome positive (Ph+) acute myeloid leukemia (Ph+AML) is discussed to be a new provisional entity for the upcoming WHO classification. Whether Ph+AML represents a distinct entity or rather embodies chronic myeloid leukemia in myeloid blast crisis (CML-BC) without preceding clinical manifestation is under debate mostly due to lack of robust criteria to reliably differentiate these two diseases. Further, while Ph+AML is clearly distinguishable from Ph+ acute lymphoblastic leukemia (Ph+ALL) based on immunophenotyping, recent studies demonstrated that Ph+AML retain typical characteristics of lymphoid disease.AimPh+AML, CML-BC and Ph+ALL were analyzed by a panel of 24 genes and array CGH to get more insights into these Ph+ leukemias and to potentially define delimiting genetic features.Patients and MethodsWe examined 24 pts with Ph+AML (11 females/13 males, median age: 58 (24-83)), 11 CML-BC (7 females/4 males, median age: 60 (32-79)) and 11 Ph+ALL (7 females/4 males, median age: 67 (44-77)). AML and ALL were diagnosed according to WHO classification by morphology, MPO and flow cytometry. CML-BC all were diagnosed as CML before and treated accordingly. All cases revealed the BCR-ABL1 fusion gene. Next generation sequencing was performed for ASXL1, BCOR, CBL, CSF3R, DNMT3A, ETV6, FLT3 tyrosine kinase domain(FLT3 -TKD), IDH1/2, JAK1/2/3, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1 and ZRSR2 using the MiSeq Instrument (Illumina, San Diego, CA). Partial tandem duplications in MLL (MLL- PTD), internal tandem duplications in FLT3 (FLT3- ITD)and deletions in IKZF1 were analyzed by quantitative real-time PCR or genescan analysis. 45 cases were investigated by array CGH (Agilent, Waldbronn, Germany).ResultsWith respect to cytogenetic abnormalities besides Philadelphia chromosome, several unbalanced abnormalities were found in Ph+ALL (mean: 9, range: 2-31), while less aberrations were found in Ph+AML and CML-BC (mean: 4, range: 0-28 and mean: 4, range: 0-16, respectively; p=0.03). Of these the most prominent aberrations which were present in all three groups included loss of 7p encompassing IKZF1 (Ph+AML: 7/23, 30%; Ph+ALL: 8/11, 73%; CML-BC: 2/10, 20%), loss of 9p encoding CDKN2A/B (Ph+AML: 2/23, 9%; Ph+ALL: 6/11, 55%; CML-BC: 2/10, 20%) as well as gain of 8q (Ph+AML: 6/23, 26%; Ph+ALL: 3/11, 27%; CML-BC:4/11, 36%). Loss of 5q (5/23, 22%), gain of 13q (4/23, 17%) and loss of 21q (3/23, 9%) was exclusively present in Ph+AML. While in Ph+ALL loss of 10q (3/11, 27%), 2p (3/11, 18%), 11q (3/11, 18%) and gain of 4q (3/11, 18%) was exclusively found. Regarding recurrent balanced aberrations no rearrangements were found in Ph+AML and Ph+ALL, while 3/11 (27%) CML-BC pts harbored balanced 3q26-rearrangements.With respect to molecular genetics, alterations were found in 15/24 (63%) Ph+AML, 8/11 (73%) CML-BC and 8/11 (73%) Ph+ALL pts. Commonly shared molecular aberrations were deletions in IKZF1 (Ph+AML: 3/24, 13%; Ph+ALL: 8/11, 73%; CML-BC: 2/10, 20%) as well as mutations (mut) in RUNX1 (Ph+AML: 5/19, 26%; Ph+ALL: 1/7, 14%; CML-BC: 5/10, 50%). Further, mut found in Ph+AML and CML-BC affected ASXL1 (Ph+AML: 2/22, 9%; CML-BC: 2/11, 18%) and IDH1 (Ph+AML: 2/22, 9%; CML-BC: 1/11, 9%). Additionally, Ph+AML harbored alterations in TP53 (3/21, 14%), TET2 (2/21, 10%) and DNMT3A (1/20, 5%). For CML-BC, additional mut were found in WT1 (2/9, 22%), ETV6 (1/9, 11%) and KRAS (1/9, 11%). Regarding FLT3 -ITD, NPM1 and the remaining genes no alterations were found. Overall, Ph+ALL differed from the combined cohort of Ph+AML and CML-BC in that mut in ASXL1, DNMT3A, ETV6, IDH1, KRAS, TET2 and WT1 as well as MLL -PTD occurred not in the former but only in the latter (12/31 cases with at least one gene mutated, p=0.07). Intriguingly, the mean±SD number of mut in these genes did not significantly differ between Ph+AML and CML-BC cases (0.36±0.58 vs. 0.67±0.71, p=0.23).ConclusionComparing cytogenetic alterations Ph+AML could be clearly distinguished from CML-BC or Ph+ALL by harboring loss of 5q and gain of 13q, which are typically found in myeloid diseases. Beside, Ph+AML and CML-BC showed a high frequency of molecular mutations which were hardly found in Ph+ALL. This data supports the concept discussed by the WHO that Ph+AML is a specific entity and can be distinguished from CML-BC and Ph+ALL. However, further studies are warranted to define the most appropriate parameters to distinguish Ph+AML from CML-BC. DisclosuresWeber:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Synergistic Effects of Combined Targeting of Beta-Catenin/CBP and Bcr-Abl in CML Blast Crisis Stem/Progenitor Cells in Vitro and in Vivo as Analyzed By Single Cell Mass Cytometry (CyTOF)

Tyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of CML. However, these agents are not able to eliminate leukemia stem cells (LSC). Additionally, TKIs have limited activity in CML blast crisis (BC). Accumulating evidence demonstrates that activation of β-catenin plays a critical role in the clonal evolution from CML chronic phase to BC and development/maintenance of CML-BC LSC. We previously demonstrated that combinations of β-catenin/CBP modulator C-82 with TKIs eradicated CD34+ CD38-/+ and quiescent TKI-resistant CML-BC stem/progenitor cells and diminished mesenchymal stem cell-mediated microenvironmental protection in vitro (Blood, 2014, 124:401). In this study, aided with single-cell mass cytometry (CyTOF) analysis and data quantification/visualization with SPADE and viSNE, we investigated the effects of combining C82 or PRI-724 (a C82 pro-drug and first-in-class β-catenin/CBP modulator) with nilotinib in various phenotypically defined CML stem/progenitor cells in vitro or in vivo in human CML-BC xenografted NOD/SCID/IL2γnull (NSG) mice.Cells from heavily-treated TKI-resistant CML-BC patients were stained with multiple cell surface markers including those defining typical LSC phenotype and intracellular survival signaling molecules and subjected to CyTOF analysis. β-catenin expression was found to be increased in various CML-BC stem/progenitor cell compartments and was highest in the CD34+ CD38+ CD123+ Tim3+ (Tim-3+ GMP) stem/progenitor subset. viSNE visualization demonstrated that overexpression of β-catenin was associated with high levels of p-CRKL, c-Myc, p-AKT, p-Tyr, p-STAT3, and p-STAT5 in CML-BC progenitor/stem cells. Cells from TKI-resistant primary CML-BC samples were then treated with C82, nilotinib, or both for 48 hours in vitro and analyzed by CyTOF. Nilotinib, as expected, had no effect on CML stem/progenitor cells in these samples. viSNE 3D visualization and quantified SPADE tree analysis revealed that C82 and more significantly the combined regimen decreased various stem/progenitor cells, especially Tim-3+ GMP cells, a subset representing the candidate for CML-BC LSC. Next, NSG mice engrafted with human CML-BCT315I/E255V cells were treated daily with PRI-724 (ALZET pump, 30 mg/kg), nilotinib (oral gavage, 50 mg/kg), or combination. At the end of a 4-week treatment, bone marrow cells were analyzed by CyTOF. viSNE 4D visualization revealed that PRI-724 and the combination strikingly reduced various CML-BC stem/progenitor subsets. SPADE tree quantification analysis demonstrated that the combination, more profoundly than PRI-724 alone, reduced the leukemia burden, CD34+ CD38-, CD34+ CD38+ ,CD34+ CD38+ CD123+, and especially Tim-3+ GMP stem/progenitor cells in NSG mice, which was further confirmed by conventional flow cytometry. Furthermore, PRI-724 alone and the combination with nilotinib induced CD11b expression in various CML-BC stem/progenitor subsets, suggesting CML-BC stem/progenitor cell differentiation. Notably, these treatments significantly inhibited CD44, a β-catenin downstream target gene and an important component in the leukemia/bone marrow niche interaction in CD34+ CD38+ CD123high and CD34+ CD38+ CD123high Tim-3high subsets; and Tim-3, a novel marker for myeloid LSC in the CD34+ CD38+ CD123high Tim-3high subset; respectively. Importantly, PRI-724 (P <0.05) and the PRI-724/nilotinib combination (P <0.01) significantly prolonged the overall survival of CML-BC-carrying NSG mice, as compared to control and the nilotinib only treated groups.Collectively, results show that inhibition of β-catenin effectively targets CML-BC stem/progenitor cells and improves survival of NSG mice engrafted with human CML-BC cells. The combination of β-catenin and tyrosine kinase inhibitors markedly enhanced the effect even in cells resistant to TKIs. Furthermore, the combination induces differentiation, and may also interrupt the interaction of CML-BC LSC with the bone marrow niche. This study suggests that combined inhibition of β-catenin and Bcr-Abl represents a novel potential strategy for targeting CML-BC stem/progenitor cells and improving patient outcomes in CML-BC that merits further investigation in preclinical and clinical settings. DisclosuresCarter:PrismBiolab: Research Funding.

Reversion to an Embryonic Alternative Splicing Program Enhances Leukemia Stem Cell Self-Renewal

BackgroundFormative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established.MethodsWhole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013).ResultsWe performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance.ConclusionIn summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication. DisclosuresJamieson:Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.