Subset Of Acute Lymphoblastic Leukemia Research Articles

Systematically Interrogating Downstream Regulation Mechanisms of HOXA9 in MLL-r Leukemia Via Functional Noncoding CRISPR Screens

HOXA9 overexpression is observed in 50-70% of human acute myeloid leukemia (AML) and a subset of acute lymphoblastic leukemia (ALL) and positively correlates with poor patient outcomes. Leukemia subtypes with hallmark overexpression of HOXA9 include those carrying MLL gene rearrangements (MLL-r), NPM1c mutations, and other genetic alterations. Frequent genetic translation of NUP98-HOXA9 retained the DNA binding domain of HOXA9, suggesting the transcription factor function is essential for leukemia initiation and maintenance. Accumulating evidence indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation. However, HOXA9 protein itself is a poor therapeutic target as it lacks targetable binding domains. Therefore, understanding HOXA9's functional downstream targets will provide alternative therapeutic targets. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory sequences and controls the downstream genes in MLL-r leukemia and across leukemias of other genetic subtypes. In this study, we have successfully established a HOXA9-miniAID expressing MLL-r B-ALL cell line to execute acute HOXA9 degradation in the same isogenic cell line background upon auxin treatment. Compared with conventional loss-of-function strategies, our AID cellular model allows the investigation of immediate downstream targets controlled by HOXA9. We have therefore identified about 1,800 reproducible peaks bound by HOXA9 at a genome-wide scale. About 80% of HOXA9 binding peaks were co-bound by HOXA9's binding partner MEIS1, and motif enrichment confirmed that more than 75% of peaks contained a typical HOXA9 consensus motif. Interestingly, >80% binding peaks are located in non-promoter cis-regulator elements. Given the essential role of HOXA9 in regulating MLL-r leukemia cell survival and differentiation, we reasoned that targeting specific HOXA9-bound peaks will affect downstream gene expression leading to a similar survival crisis seen in HOXA9 knockout cells. Based on this rationale, combinatorial CRISPR screens were performed in three biological replicates on the MLL-r SEM cell line stably expressing Cas9 and base editor (ABE8.0), followed by time-course dropout selection on days 7, 14, and 21. Integrative CRISPR screen analysis identified six reproducible noncoding hits, including a positive control located in the distal enhancer of FLT3. Functional validation by targeting these six noncoding segments with Cas9, dCas9-KRAB in additional MLL-r leukemia cell lines MOLM13, MV4,11 confirmed the survival essential of these HOXA9-bound regions. No cellular phenotype was observed in non-MLL-r leukemia cell lines, including NALM6, JURKAT, or OCI-AML2. Transplanting SEM cells targeted with sgHOXA9-in-FLT3 into NSG mice, significant growth retardation was monitored by flow analysis of peripheral blood. In addition, RNA-seq and Q-PCR analysis further identified the functional relevant downstream genes upon CRISPR editing of these HOXA9-bound noncoding segments, including FLT3, XBP1, JUN, BAHCC1, and CCDC200. Chromatin conformation characterization by Capture-C further confirmed the long-range interaction between HOXA9 bound enhancers and corresponding gene promoters in MLL-r SEM cells. In summary, using state-of-art research tools and newly established cell models, we have conducted a systematic functional screen and mechanism study of an undocumented downstream transcriptional network of HOXA9 in MLL-r leukemia cells. The work is significant as it will advance our understanding of how HOXA9-associated transcription programs reconstruct the regulatory network specifying MLL-r dependency. Moreover, our study will promote the future development of alternative therapeutic targets in HOXA9-driven (including HOXA9 and possibly NUP98-HOXA9 subtype) leukemia in patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pooled analysis of recent studies of magnetic fields and childhood leukemia

BackgroundOver forty epidemiologic studies have addressed an association between measured or calculated extremely-low-frequency magnetic fields (MF) and childhood leukemia. These studies have been aggregated in a series of pooled analyses, but it has been 10 years since the last such. MethodsWe present a pooled analysis combining individual-level data (24,994 cases, 30,769 controls) from four recent studies on MF and childhood leukemia. ResultsUnlike previous pooled analyses, we found no increased risk of leukemia among children exposed to greater MF: odds ratio (OR) = 1.01, for exposure ≥0.4 μT (μT) compared with exposures <0.1 μT. Similarly, no association was observed in the subset of acute lymphoblastic leukemia, birth homes, studies using calculated fields, or when geocoding accuracy was ignored. In these studies, there is a decline in risk over time, also evident when we compare three pooled analyses. A meta-analysis of the three pooled analyses overall presents an OR of 1.45 (95% CI: 0.95–2.20) for exposures ≥0.4 μT. ConclusionsOur results are not in line with previous pooled analysis and show a decrease in effect to no association between MF and childhood leukemia. This could be due to methodological issues, random chance, or a true finding of disappearing effect.

Therapy-related acute lymphoblastic leukemia is a distinct entity with adverse genetic features and clinical outcomes

AbstractPatients with therapy-related acute lymphoblastic leukemia (t-ALL) represent a small subset of acute lymphoblastic leukemia (ALL) patients who received genotoxic therapy (ie, chemotherapy or radiation) for a prior malignancy. These patients should be distinguished from patients with de novo ALL (dn-ALL) and ALL patients who have a history of prior malignancy but have not received cytotoxic therapies in the past (acute lymphoblastic leukemia with prior malignancy [pm-ALL]). We report a retrospective multi-institutional study of patients with t-ALL (n = 116), dn-ALL (n = 100), and pm-ALL (n = 20) to investigate the impact of prior cytotoxic therapies on clinical outcomes. Compared with patients with pm-ALL, t-ALL patients had a significantly shorter interval between the first malignancy and ALL diagnosis and a higher frequency of poor-risk cytogenetic features, including KMT2A rearrangements and myelodysplastic syndrome-like abnormalities (eg, monosomal karyotype). We observed a variety of mutations among t-ALL patients, with the majority of patients exhibiting mutations that were more common with myeloid malignancies (eg, DNMT3A, RUNX1, ASXL1), whereas others had ALL-type mutations (eg, CDKN2A, IKZF1). Median overall survival was significantly shorter in the t-ALL cohort compared with patients with dn-ALL or pm-ALL. Patients who were eligible for hematopoietic cell transplantation had improved long-term survival. Collectively, our results support t-ALL as a distinct entity based on its biologic and clinical features.

Abstract 5820: Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset

Abstract While intensive chemotherapy regimens result in remission in approximately 80% of pediatric and 45% of adult ALL patients, new approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Early T-cell precursor (ETP) ALL, a subset of T-ALL associated with a higher rate of relapse and induction failure, with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. PIM protein kinases are overexpressed in a number of hematopoietic malignancies, including ALL, with its overexpression being associated with poor prognosis. Pan-PIM inhibitors (PIMi) are being tested in clinical trials in patients with relapsed/refractory multiple myeloma and acute myeloid leukemia. Analysis of the effects of PIMi (AZD1208/LGB321) on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Immunophenotype of all the PIMi insensitive cells lines was consistent with a more mature T-ALL phenotype. In contrast, sensitive cells, had an immature or ETP-ALL phenotype. Bioinformatics analysis of three independent T-ALL patient datasets (GSE28703, E-MEXP-313, and GSE62156) demonstrated overexpression of PIM1 in the majority of ETP-ALLs and in a small subset of non-ETP-ALL. We also identified 58 genes changing significantly between PIMi sensitive versus insensitive cell lines that were common to the differential genes between ETP versus non-ETP ALL cases from GSE28703, suggesting that high PIM1 expression along with 58 gene signature could be a driver of the ETP phenotype. Importantly, we have made the novel observation that, combination of Ponatinib, a tyrosine kinase inhibitor (TKI) with the PIMi is synergistically lethal to T-ALL cells with ETP phenotype. These agents appear to work together by inducing PARP and Caspase cleavage, sufficient to drive apoptosis. Following engraftment of NSG mice with H-SB2-Luc cells, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly abrogated leukemia as evidenced by optical scanning for luciferase cells, and reduced number of hCD45+ leukemic cells in the peripheral blood and bone marrow. Also, the dual therapy significantly prolonged the survival of the treated mice. In summary, our work demonstrates a strong preclinical rationale for a novel treatment strategy of combining PIMi and TKI to treat T-ALL patients with high PIM expression. Citation Format: Sathish Kumar Reddy Padi, Libia Luevano, Neha Singh, Jiin Song, Ritu Pandey, Jon C. Aster, Xue-Zhong Yu, Shikhar Mehrotra, Andrew Kraft. Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5820. doi:10.1158/1538-7445.AM2017-5820

Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset.

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

Illegitimate RAG-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia.

Breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1), encoded by the Philadelphia (Ph) chromosome, is the characteristic of chronic myeloid leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). We demonstrated that expression of the Ik6 transcript, which lacked exons 3-6, was observed exclusively in BCR-ABL1(+) B ALL and lymphoid blast crisis CML (BC-CML) patients harbouring the IKZF1 Δ3-6 deletion. To confirm the hypothesis that illegitimate recombination activating gene protein (RAG)-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia, we first demonstrated that the expression rates of RAG1 and RAG2, collectively called RAG, were higher in ALL and BC-CML (lymphoid). Notably, analysis of relationships among RAG, BCR-ABL1 and Ikaros 6 (Ik6) showed that Ik6 can be generated only if RAG and BCR-ABL1 are co-existing. The sequencing data showed that the deleted segments of introns 2 and 6 contained cryptic recombination signal sequences (cRSSs) and frequently had non-template nucleotides inserted between breakpoints. Furthermore, we used chromatin immunoprecipitation (ChIP) technology and demonstrated that the sequences directly flanking IKZF1 Δ3-6 deletion breakpoints have significantly higher levels of histone H3 lysine 4 trimethylation (H3K4me3) modifications. Overall, RAG expression, good-quality cRSS and a specific chromatin modification, H3K4me3, satisfy the conditions of RAG's off-target effects on IKZF1. Our work provides evidence for RAG-mediated IKZF1 Δ3-6 deletion. Our results raise the prospect that RAG is a valuable biomarker in disease surveillance. Dissecting the contribution of RAG should not only provide valuable mechanistic insights, but will also lead to a new therapeutic direction.

Genetic Heterogeneity of Philadelphia Chromosome-Positive Leukemias With a Spotlight on Two Cases: Relapsed Acute Lymphoblastic Leukemia and De Novo Acute Myeloid Leukemia

The Philadelphia chromosome (Ph) resulting from t(9;22)(q34;q11) is the hallmark of chronic myeloid leukemia (CML), and is also identified in a subset of acute lymphoblastic leukemia (ALL) and rarely in acute myeloid leukemia (AML). This derivative chromosome contains the BCR-ABL1 fusion gene that encodes for a constitutively active tyrosine kinase. Amplification of BCR-ABL1 and mutations in the ABL1 kinase domain are known causes of tyrosine …

Abstract 2372: Inflammatory cytokine induced TSLP from bone marrow niches contributes to relapse of high risk TSLPR overexpressing acute lymphoblastic leukemia

Abstract Although 5 year survival rates are near 90%, leukemia is the most common cause of death from cancer in children with treatment failure primarily caused by relapse. A subset of acute lymphoblastic leukemia (ALL) patients that have an increased risk of relapse and consequently poorer overall survival have leukemia that overexpresses thymic stromal lymphopoietin receptor (TSLPR/CRLF2). However, it remains unclear what role Thymic Stromal Lymphopoietin (TLSP) signaling may play in increasing the risk of relapse. We hypothesize that TSLP/TSLPR axis may support progression of ALL by providing a signal through overexpressed TSLPR, which would sensitize the leukemia to low levels of the cytokine in bone marrow (BM) niches and promote the survival of the leukemia. We have created a high TSLPR expressing leukemia model (TSLPRhigh) through retroviral transduction of a transplantable syngeneic mouse ALL model (TSLPRlow) in which leukemia progression can be examined. This TSLPRhigh leukemia has expression of TSLPR comparable to what is found on patient derived ALL that naturally overexpress TSLPR and the receptor is functional as we see increased phosphorylation of STAT5 protein in response to TSLP stimulation. When ALL lines were injected into mice, we observed an 8 fold difference in the percentage of TSLPRhigh vs. TSLPRlow ALL in the BM 5 days after injection, where leukemia accounted for less than 4% of the BM corresponding to an early stage of ALL progression, whereas overall leukemia progression was not significantly different. This implies that the TSLP axis is most critical at low leukemic burden when bone marrow niches are intact and not disrupted by high levels of ALL in the BM. TSLPRlow and TSLPRhigh expressing cells show no significant difference in growth rate in vitro or in vivo, however when we treated ALL cells with chemotherapy in the absence or presence of TSLP we found that TSLP signaling significantly decreased the number of apoptotic cells in TSLPRhigh but not TSLPRlow ALL. These data suggest that high TSLPR expression sensitizes leukemia cells to TSLP to promote survival and not growth of ALL. This is significant because we believe that TSLP is likely to be expressed at low levels in the BM under normal circumstances. Interestingly, we have evidence using a nestin positive murine BM stromal cell line and stromal cells derived from fresh murine BM that TSLP mRNA and protein is induced by IL-1α and TNF-α stimulation. We further have found that cytarabine treatment of TSLPRhigh cells leads to induction of both IL-1α and TNF-α suggesting that chemotherapeutic treatment of ALL could potentially provide an unintended advantage to TSLPR overexpressing ALL. Based on this data, we postulate that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse in TSLPR overexpressing ALL particularly in the context of standard therapy. Citation Format: Christopher D. Chien, Sang Nguyen, Haiying Qin, Terry J. Fry. Inflammatory cytokine induced TSLP from bone marrow niches contributes to relapse of high risk TSLPR overexpressing acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2372. doi:10.1158/1538-7445.AM2015-2372

ABL001, a Potent Allosteric Inhibitor of BCR-ABL, Prevents Emergence of Resistant Disease When Administered in Combination with Nilotinib in an in Vivo Murine Model of Chronic Myeloid Leukemia

ABL001, a Potent Allosteric Inhibitor of BCR-ABL, Prevents Emergence of Resistant Disease When Administered in Combination with Nilotinib in an in Vivo Murine Model of Chronic Myeloid Leukemia

MDR1 Mediated Drug Resistance to a Histone Methyltansferase Inhibitor (KMT)

Aberrant histone methylation is emerging as a major contributor to oncogenesis, and inhibitors of histone lysine methyltransferases (KMTs) are starting to enter early clinical studies. Currently, inhibitors for two KMTs are in phase I/II clinical trials, an inhibitor of the histone 3 lysine 79 (H3K79) methyltransferase DOT1L for MLL-rearrange leukemias (NCT02141828), and inhibitors for the H3K27 methyltransferase EZH2 for lymphomas or solid tumors with activating EZH2 mutations (NCT01897571 and NCT02082977). The next few months and years are likely to see more KMT inhibitors entering clinical development, such as compounds directed against the H3K36 methyltransferase MMSET/NSD2/WHSC1, which is commonly translocated in multiple myeloma and carries activating mutations in a subset of acute lymphoblastic leukemia. Typically, KMT inhibitors are structurally similar and are derived from the methyl donor S-adenosyl-methionine (SAM). Very little is known about possible mechanisms of resistance to these compounds.We exposed a panel of leukemia cell lines to varying concentrations of the DOT1L inhibitor EPZ4777 and assessed the decrease of H3K79 di-methylation by Western Blotting. Our analysis revealed a dose-dependent decrease of H3K79 dimethylation in most cell lines. Interestingly, we identified a pair of cell lines, KG-1 and KG-1a, which were near completely resistant to EPZ4777 mediated DOT1L inhibition, with no effect of 10 μM EPZ4777 (4 day exposure). Even after exposure to EPZ4777 for 15 days, KG-1 showed only a slight decrease in H3K79me2 marking and no decrease in marking was seen for KG-1a up to a concentration of 3 μM. We searched the encode database for expression levels of DOT1L, as well as higher expression of proteins previously described as mediating drug resistance in AML cell lines. We found no significantly higher DOT1L expression level, suggesting that overexpression of the target is not the predominant mechanisms of resistance in KG-1/KG-1a. We confirmed similar baseline H3K79 methylation levels in KG1/KG1a and the other cell lines in our panel by Western Blotting. However, we noted a higher expression of multidrug resistance protein 1 (MDR1/p-glycoprotein/ABCB1) in the KG-1 and KG-1a cell lines compared to the other cell lines in our panel. To explore whether DOT1L inhibition by EPZ4777 could be restored in KG1 and KG1a cells when MDR1 was inhibited, cells were exposed to 0.3 μM EPZ4777 and varying concentrations of Cyclosporine A (CSA), a known inhibitor of MDR1. Increasing concentration of CSA with a fixed dose of 0.3 μM EPZ4777 led to a dose dependent decrease in H3K79me2 in KG-1/KG-1a, suggesting that inhibition of MDR1 increased the intracellularly effective dose of EPZ4777. CSA at a concentration of 0.3 μM and 0.3 μM EPZ4777 near completely abolished H3K79 methylation. In summary, our results suggest that KMT inhibitors can be subject to classic drug resistance mechanisms such as the MDR1/p-glycoprotein/ABCB1 efflux pump. These data provide insight for the use and design of small-molecule inhibitors for KMTs. DisclosuresNo relevant conflicts of interest to declare.

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.

Abstract SY19-04: Design and development of ponatinib, a pan-BCR-ABL inhibitor for CML.

Abstract SY19-04: Design and development of ponatinib, a pan-BCR-ABL inhibitor for CML.

The Growing Arsenal of ATP-Competitive and Allosteric Inhibitors of BCR–ABL

The BCR-ABL fusion kinase is the driving mutation of chronic myelogenous leukemias and is also expressed in a subset of acute lymphoblastic leukemias. Recent advances in elucidating the structure, regulation, and signaling of BCR-ABL have led to the identification of allosteric sites that are distant from the ATP-binding pocket and are critical for BCR-ABL-dependent oncogenic transformation. Here, we review the available data regarding the molecular mechanism of action and the specificity of ATP-competitive tyrosine kinase inhibitors targeting BCR-ABL. In addition, we discuss how targeting of allosteric sites could provide new opportunities to inhibit resistant BCR-ABL mutants, either alone or in combination with conventional ATP-competitive inhibitors.

Targeting BCR-ABL Oncoprotein for Leukemia Therapy: Current Biotechnology and Future Perspectives

The chimeric oncoprotein BCR-ABL, resulting from Philadelphia (Ph) chromosome translocation, is crucial for the pathogenesis of chronic myelogenous leukemia (CML) as well as a subset of acute lymphoblastic leukemia (ALL). Due to the loss of regulatory motifs during the fusion process, BCR-ABL has constitutive tyrosine kinase activity that is critical for oncogenesis. This provides the rationale for developing drugs that specifically inhibit BCR-ABL tyrosine kinase activity. The first tyrosine kinase inhibitor (TKI), imatinib mesylate (STI571, Gleevec®), was launched in 2001, completely changing the landscape of therapy for CML. However, imatinib-resistant cases emerged in the clinic, most caused by mutations in the BCR-ABL tyrosine kinase domain or BCR-ABL gene amplification. This urged the development of next-generation TKIs that can override imatinib-resistance. Instead of inhibiting tyrosine kinase activity, an alternative strategy, currently being tested in clinical trials, is to induce BCR-ABL degradation by heat shock protein 90 inhibitors or proteasome inhibitors. A more tentative strategy is to entrap BCR-ABL in the nucleus based on the finding that nuclear BCR-ABL is pro-apoptotic. Moreover, the unique mRNA and amino acid sequences at the junctional region of BCR-ABL provide an opportunity for specific targeting by gene therapy and immunotherapy, respectively. Therefore, a deep understanding of the biology of BCR-ABL in the context of CML and the use of state-of-the-art biotechnology have aided and will continue to aid in the development of targeted therapy for BCR-ABL-driven leukemia and minimal residue disease (MRD). Keywords: Antisense therapy, ALL, BCR-ABL, CML, HSP90 inhibitor, imatinib, imatinib-resistance, leukemia vaccine, minimal residual disease, nuclear entrapment, proteasome inhibitor, ribozyme, RNA interference, T315I mutation, tyrosine kinase inhibitor.

Fine structure of translocation breakpoints within the major breakpoint region in BCR-ABL1-positive leukemias

The chromosomal translocation t(9;22)(q34;q22), with expression of the BCR-ABL1 fusion gene is the cytogenetic and molecular hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). Basically two types of BCR-ABL1 chimeric mRNA transcripts have been observed: (1) e13a2/e14a2 transcripts in CML and ALL, resulting from chromosomal breaks in the major breakpoint cluster region (M-bcr) of the BCR gene and (2) e1a2 transcripts in ALL resulting from breaks in the minor breakpoint cluster region (m-bcr) of the BCR gene. To gain a better understanding of this molecular alteration, we developed a long-distance inverse PCR (LDI PCR) method for M-bcr breakpoint identification in BCR-ABL1-positive cases and were thus able to identify the chromosomal breakpoints within the M-bcr in 62 BCR-ABL1-positive samples. The corresponding reciprocal breakpoints were identified and molecularly characterized in 45 of these cases. In 2 samples, the breaks were located 5′ to the ABL1 locus and in one case, the der(9) break was identified on 9q34.13 several hundred kB 3′ telomeric to ABL1. The analysis of breaks revealed no significant clustering and no association with repetitive elements (Alu, L1, L2) or recombination signal sequence sites. The established LDI PCR permits fast, relatively easy and unbiased identification of breakpoints in the M-bcr region of BCR and also enables the molecular analysis of more complex translocations with breakpoints outside the ABL1 gene locus or other BCR fusion genes.

Allogeneic hematopoietic stem cell transplantation overcomes the adverse prognostic impact of CD20 expression in acute lymphoblastic leukemia

AbstractCD20 expression is associated with early recurrence and inferior survival in precursor-B acute lymphoblastic leukemia patients treated with chemotherapy. Whether CD20 influences outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unknown. We analyzed CD20 expression on blasts at diagnosis in 157 patients who underwent allo-HSCT in the first complete remission (57%) or the second complete remission (43%). Of 125 evaluable patients, 71 were ≥ 20 years of age. CD20 expression was observed in 58 patients (46%; 52% of children, 39% of adults). There was no association between age, Ph+ status, white blood cell count at diagnosis, and CD20 positivity. After allo-HSCT, disease-free survival at 5 years was 48% for all patients, 55% (95% confidence interval 40%-67%) for CD20+ patients, and 43% (95% confidence interval 30%-54%) for CD20− patients (P = .15). Relapse did not differ between the groups. These results can serve as a reference to evaluate incorporation of anti-CD20 therapeutics to HSCT for the CD20+ acute lymphoblastic leukemia subset. Clinical trial numbers for www.clinicaltrials.gov are NCT00365287, NCT00305682, and NCT00303719.

Mutation-specific control of BCR-ABL T315I positive leukemia with a recombinant yeast-based therapeutic vaccine in a murine model

Chromosomal translocations generating the BCR-ABL oncogene cause chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia. The BCR-ABL T315I mutation confers drug resistance to FDA-approved targeted therapeutics imatinib mesylate, dasatinib, and nilotinib. We tested the ability of a recombinant yeast-based vaccine expressing the T315I-mutated BCR-ABL antigen to stimulate an anti-BCR-ABL T315I immune response. The yeast-based immunotherapy significantly reduced or eliminated BCR-ABL T315I leukemia cells from the peripheral blood of immunized animals and extended leukemia-free survival in a murine model of BCR-ABL + leukemia compared to animals receiving sham injection or yeast expressing ovalbumin. With immunization, leukemic cells harboring BCR-ABL T315I were selectively eliminated after challenge with a mixed population of BCR-ABL and BCR-ABL T315I leukemias. In summary, yeast-based immunotherapy represents a novel approach against the emergence of cancer drug resistance by the pre-emptive targeted ablation of tumor escape mutants.

Bruton's tyrosine kinase is not essential for Bcr-Abl-mediated transformation of lymphoid or myeloid cells

Bcr-Abl, a constitutively active tyrosine kinase, is the cause of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemias (ALL). Bruton's tyrosine kinase (BTK), a member of the Tec family of tyrosine kinases with a crucial role in B-cell development, is consistently tyrosine phosphorylated in Bcr-Abl expressing murine pre B cells. BTK has been implicated in Bcr-Abl-mediated B-cell transformation and resistance to imatinib, implying that inhibiting BTK may be therapeutically beneficial. We decided to test whether BTK is a critical node in Bcr-Abl transformation and potential drug target in imatinib-resistant Bcr-Abl-positive cells. We depleted BTK in Ba/F3 and 32D cells expressing native and kinase domain (KD) mutant (E255K and T315I) Bcr-Abl, using shRNA. BTK levels were reduced to <10% of controls. However, no differences in viability and cell proliferation were observed and the response to imatinib was not altered. Consistent with this, proliferation and viability were unaffected by inhibition of BTK with reversible (PC-005) and irreversible (PCI-31523) small molecule inhibitors. Lastly, BTK inhibition did not affect the ability of Bcr-Abl to transform primary murine hematopoietic cells in colony forming and B-cell transformation assays. Collectively this data argues against a critical role for BTK in Bcr-Abl-mediated leukemogenesis.

BCR–ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros

The Philadelphia chromosome, a chromosomal abnormality that encodes BCR-ABL1, is the defining lesion of chronic myelogenous leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). To define oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we performed a genome-wide analysis of diagnostic leukaemia samples from 304 individuals with ALL, including 43 BCR-ABL1 B-progenitor ALLs and 23 CML cases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR-ABL1 ALL, but not in chronic-phase CML. Deletion of IKZF1 was also identified as an acquired lesion at the time of transformation of CML to ALL (lymphoid blast crisis). The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant-negative Ikaros isoform, or the complete loss of Ikaros expression. Sequencing of IKZF1 deletion breakpoints suggested that aberrant RAG-mediated recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are an important event in the development of BCR-ABL1 ALL.

Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1–transformed acute lymphoblastic leukemia cells

The Philadelphia chromosome (Ph) encoding the oncogenic BCR-ABL1 kinase defines a subset of acute lymphoblastic leukemia (ALL) with a particularly unfavorable prognosis. ALL cells are derived from B cell precursors in most cases and typically carry rearranged immunoglobulin heavy chain (IGH) variable (V) region genes devoid of somatic mutations. Somatic hypermutation is restricted to mature germinal center B cells and depends on activation-induced cytidine deaminase (AID). Studying AID expression in 108 cases of ALL, we detected AID mRNA in 24 of 28 Ph+ ALLs as compared with 6 of 80 Ph− ALLs. Forced expression of BCR-ABL1 in Ph− ALL cells and inhibition of the BCR-ABL1 kinase showed that aberrant expression of AID depends on BCR-ABL1 kinase activity. Consistent with aberrant AID expression in Ph+ ALL, IGH V region genes and BCL6 were mutated in many Ph+ but unmutated in most Ph− cases. In addition, AID introduced DNA single-strand breaks within the tumor suppressor gene CDKN2B in Ph+ ALL cells, which was sensitive to BCR-ABL1 kinase inhibition and silencing of AID expression by RNA interference. These findings identify AID as a BCR-ABL1–induced mutator in Ph+ ALL cells, which may be relevant with respect to the particularly unfavorable prognosis of this leukemia subset.