B-progenitor Acute Lymphoblastic Leukemia Research Articles

Enhancer retargeting of CDX2 and UBTF::ATXN7L3 define a subtype of high-risk B-progenitor acute lymphoblastic leukemia

AbstractTranscriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required.

Clinical Effect of Genetic Alterations in Pediatric Patients with B-Progenitor Acute Lymphoblastic Leukemia

▪BackgroundRecent genetic studies using high-throughput sequencing have substantially revealed genetic alterations in pediatric patients with B-progenitor acute lymphoblastic leukemia (B-ALL). However, their clinical relevance of these alterations has not been fully investigated.MethodsWe performed targeted-capture sequencing of 149 known or putative driver genes for B-ALL in 525 pediatric patients who were uniformly treated according to the Japan Association of Childhood Leukemia Study ALL-02 protocol. In addition, we performed whole transcriptome sequencing in 63 of 118 patients who were not categorized into any genetic subgroup defined by RT-PCR status or targeted-capture sequencing results.ResultsIn total, 647 focal deletions (median 1 per patient; range, 0-7) and 993 mutations (median 2 per patient; range, 0-10) were detected in 491 (94%) cases. Some of these alterations were closely associated with disease subtypes. We identified recurrent PHF6 mutations, which were almost exclusively detected in the TCF3 - PBX1- positive ALL genotype. Furthermore, frequent involvement of PAX5 and KMT2D (mutations) and IKZF3 (deletions) were characteristic of TCF3 - PBX1 -positive ALL genotype. To identify genetic alterations associated with a poor prognosis, we analyzed 20 genes that were mutated in at least 20 patients in our study cohort. Univariate analyses revealed genetic alterations in TP53 , KMT2D , and IKZF1 to be associated with a significantly shorter overall survival. Mutations and deletions in IKZF1 were also strongly associated with induction failure (Odds ratio, 12; 95% CI, 2.7-55). This led to death in 80% (n = 4/5) of the patients with IKZF1 alterations. Most patients with TP53 and KMT2D mutations and with disease relapse had fatal disease progression [94% mortality rate (n = 17/18)]. Multivariate analysis, together with the NCI criteria, revealed that two genetic lesions, TP53 and KMT2D , retained prognostic impacts for overall survival. Prediction of 5-year OS improved in each NCI-risk group when patients in each risk group were stratified according to the presence or absence of mutations in TP53 or KMT2D lesions: 69% [with alteration (n = 26)] vs. 85% [without alteration (n = 138)] for the NCI-HR group (P = 0.03) and 85% [with alteration (n = 42)] vs. 97% [without alteration (n = 319)] for the NCI-SR group (P = 0.004). Additionally, the four less frequently mutated genes also had an independent prognostic impact before and after adjustment for risk factors: IKZF2 (n = 6; HR for death, 7.4; 95% CI, 2.3-24), SH2B3 (n = 4; HR for death, 7.0; 95% CI, 1.7-29), JAK2 (n = 7; HR for events, 4.6; 95% CI, 1.7-12), and PTEN (n = 9; HR for death, 3.8; 95% CI, 1.2-12). Notably, mutations in three genes present potentially druggable targets: ruxolitinib for mutations in JAK2 or SH2B3 and poly (ADP-ribose) polymerase inhibitor formutations in PTEN . Finally, based on a gene expression profile, 20 of 63 patients were categorized into discrete subgroups characterized by Ph-like profiles (n = 7), ZNF384 (n = 7), and MEF2D (n = 6) fusions. Ph-like and MEF2D fusion-positive ALLs were associated with a dismal prognosis, whereas all patients with a ZNF384 fusion survived with complete remission. No mutations were associated with a poor prognosis in the 43 remaining patients. Further analysis, such as whole genome sequencing, is warranted for these patients.ConclusionsTargeted-capture sequencing improves the prediction of clinical outcomes and helps detect prognostic and druggable biomarkers. Our results can facilitate the clinical applications of next-generation sequencing to select optimal therapy for pediatric patients with B-ALL. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Clinical Significance of Phd Finger Protein 2 As a Novel Ikaros Target in Acute Lymphoblastic Leukemia

Background: PHD finger protein 2 (PHF2) mediates demethylation of dimethylated ‘Lys-9‘ of histone H3 (H3K9me2), followed by transcription activation of target genes. PHF2 is a positive epigenetic regulator and is associated with tumor suppression in several types of cancer. Until now, there have been no reports about the PHF2 expression in leukemia patients and its roles in acute lymphoblastic leukemia (ALL).Genetic alterations of IKZF1 encoding the lymphoid transcription factor IKAROS are a hallmark of high risk B-progenitor ALL and are associated with poor outcome. However, it is still undetermined whether and how IKAROS regulates PHF2 expression. Methods: The 164 patients with newly-diagnosed ALL (107 B-ALL and 57 T-ALL; 12-77 years old) were recruited between June 2008 and June 2016 at the First Affiliated Hospital of Nanjing Medical University and Zhongda Hospital Southeast University. The study was approved by the Ethics Committee of the two hospitals. Patients were allocated into high or low PHF2 expression cohorts (4th quartile vs . 1st-3rd quartiles) with a cut-off value determined by SPSS 20.0. The genomic amplification combined direct sequencing was used for detection of IKZF1 deletion. PHF2 expression was determined by qPCR in cells . The enrichment of IKAROS binding and H3K4me3 in the promoter of PHF2 was determined by the chromatin-immunoprecipitation assay combined qPCR (qChIP). IKZF1 knockdown was achieved in cells by transfection of human IKZF1 shRNA constructs in the GFP vector using the Neon Transfection System and with scrambled shRNA as a control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using univariate and multivariate Cox models. Relapse-free survival and overall survival were estimated by the Kaplan-Meier method and compared by log-rank test. Statistical analyses used SPSS version 20.0. Results: PHF2 mRNA levels in bone marrow samples from adults with ALL, especially those with B-cell ALL (B-ALL), were significantly lower than those in normals. The B-ALL cohort with low PHF2 mRNA levels was associated with a higher median bone marrow blast percentage (90.4% vs. 82.4%; P=0.001), a higher percentage of stem cell marker CD34+ (82.3% vs. 60.0%; P=0.021), myeloid marker CD33+ (48.5% vs. 25.0%; P=0.046), splenomegaly (46.8% vs. 23.1%, P=0.034) and delayed remission more than 4 weeks (48.7% vs. 19.0%; P=0.015), also a higher frequency of Ik6 (+) (46.3% vs. 8.0%, P=0.001) and a lower median PLT count in comparison to the high PHF2 expression cohort. Moreover, ChIP-seq data identified IKAROS binding peaks in the PHF2 promoter region in Nalm6 B-ALL and primary B-ALL cellsand the bindings were confirmed by the qChIP assay . IKAROS overexpression promotes PHF2 mRNA levels, conversely, IKZF1 knockdown decreased PHF2 expression in Nalm6 and CEM cells. These data indicate a direct effect of IKAROS on PHF2 transcription. The patients with IKZF1 deletion (Ik 6) have significantly lower PHF2 expression (0.03201 ± 0.000527 vs. 0.11331± 0.05773; P=0.033), which suggests that the IKZF1 deletion may contribute to low PHF2 expression in ALL patients. Treatment of Nalm6 and CEM cells with CK2 inhibitor CX-4945, which functions as IKAROS activator, promoted expression of PHF2 mRNA in ALL cell lines and primary cells and IKZF1 knockdown blocks this effect. CX-4945 treatment also increases the binding of IKAROS and H3K4me3 enrichment to the PHF2 promoter region in Nalm6 and CEM ALL cells as well as in primary ALL cells. Conclusion: PHF2 low expression was significantly associated with leukemia proliferation and several poor prognostic indicators in adult ALL patients. IKAROS directly regulates PHF2 transcription, and that CK2 inhibitor promotes PHF2 transcription through histone modification in an IKAROS dependent manner. Our data demonstrates the oncogenic effects of IKAROS dysfunction induced decreases in PHF2 expression. This is the first report to demonstrate that low PHF2 expression is significantly associated with Acute Lymphoblastic Leukemia. We suggest that PHF2 expression levelsmay be integrated into future adult ALL risk-stratification models. DisclosuresDovat:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees.

Development and Validation of an Rnaseq NGS Assay to Comprehensively Detect Clinically Relevant ALL Gene Fusions

BackgroundRecent advances in molecular technologies have identified a subtype of B-progenitor acute lymphoblastic leukemia (B-ALL) with a Philadelphia chromosome-like (Ph-like or BCR-ABL1-like) gene expression profile without the BCR-ABL1 fusion. The subtypes containing Ph-like fusions are considered the more aggressive subtype. Although FISH and RT-PCR assays can be used to detect these fusions, these technologies are labor intensive and low-throughput in nature. An optimal solution is to utilize RNA-sequencing for high-throughput detection of characterized and novel fusions with high accuracy and sensitivity in a clinical setting. Here we report the development and characterization of a fusion panel targeting 115 genes of which 23 are specifically designed to detect fusions of clinical relevance in ALL. The genes targeted in the ALL panel include: ABL1, ABL2, BCR, CRLF2, CSF1R, ETV6, IL2RB, IL3, JAK2, KMT2A, MEF2D, MLLT109, NUP98, PAX5, PDGFRB, PTK2B, RUNX1, TAL1, TCF3, TLX1, TLX3, TYK2, and ZNF284.Materials & MethodsSamples (peripheral blood, bone marrow, or fixed cell pellets) were selected based on FISH or molecular analysis that contain gene fusions in the ALL panel. RNA was extracted from these samples using the Qiagen AllPrep or the MagNA Pure isolation platform. A custom ArcherDX Anchored Multiplex PCR (AMP) assay was designed to enrich for a set of oncology-related genes including ALL relevant genes using 250ng input RNA. The enriched libraries were pooled and sequenced on an Illumina NextSeq500. Analysis was performed using Archer Analysis software, version 5.1. Additional filter settings were employed to reduce the false-positive rate and increase confidence in true-positive calls. The passing fusions were parsed through Cartagenia and N-of-One clinical interpretation for annotation and patient reporting. The accuracy, specificity, analytical sensitivity, precision, and reproducibility of the RNASeq assay were evaluated.ResultsStudies utilizing clinical patient samples as well as synthetic G-block constructs containing specific clinically relevant fusion break points were performed using the custom RNA FusionPlex panel to detect the fusions. The criteria for successful fusion detection were established at 10 unique molecules supporting the fusion with 3 unique start sites. These studies clearly demonstrated that the custom RNA FusionPlex test was able to successfully detect fusions in the ALL genes contained in the fusion panel. In addition, the fusions detected included most of the relevant fusions that have been reported previously by either FISH or RT-PCR testing, demonstrating that the custom RNA FusionPlex test is effective in detecting clinically relevant fusions. The accuracy of the assay was >90%, with precision and reproducibility at >95%. The false positive rate of the assay using these criteria was observed to less than 2%. Additional metrics and criteria will also be presented.ConclusionsThe studies presented clearly demonstrate that the Genoptix custom RNA Fusionplex test is capable of detecting most clinically relevant ALL gene fusions in patient samples in a reproducible and effective manner. The Genoptix ALL panel is a highly sensitive and robust product for detection of gene fusions in a clinical setting and presents a viable alternative to traditional FISH and RT-PCR testing, capable of delivering comprehensive patient gene fusion information. DisclosuresNo relevant conflicts of interest to declare.

Tumor-intrinsic and -extrinsic determinants of response to blinatumomab in adults with B-ALL

AbstractBlinatumomab, a bispecific antibody that directs CD3+ T cells to CD19+ tumor cells, shows variable efficacy in B-progenitor acute lymphoblastic leukemia (B-ALL). To determine tumor-intrinsic and -extrinsic determinants of response, we studied 44 adults with relapsed or refractory B-ALL (including 2 minimal residual disease positive) treated with blinatumomab using bulk tumor and single-cell sequencing. The overall response rate in patients with hematological disease was 55%, with a high response rate in those with CRLF2-rearranged Philadelphia chromosome–like ALL (12 [75%] of 16). Pretreatment samples of responders exhibited a tumor-intrinsic transcriptomic signature of heightened immune response. Multiple mechanisms resulted in loss of CD19 expression, including CD19 mutations, CD19-mutant allele-specific expression, low CD19 RNA expression, and mutations in CD19 signaling complex member CD81. Patients with low hypodiploid ALL were prone to CD19− relapse resulting from aneuploidy-mediated loss of the nonmutated CD19 allele. Increased expression of a CD19 isoform with intraexonic splicing of exon 2, CD19 ex2part, at baseline or during therapy was associated with treatment failure. These analyses demonstrate both tumor-intrinsic and -extrinsic factors influence blinatumomab response. We show that CD19 mutations are commonly detected in CD19− relapse during blinatumomab treatment. Identification of the CD19 ex2part splice variant represents a new biomarker predictive of blinatumomab therapy failure.

EBF1 and Pax5 safeguard leukemic transformation by limiting IL-7 signaling, Myc expression, and folate metabolism.

EBF1 and PAX5 mutations are associated with the development of B progenitor acute lymphoblastic leukemia (B-ALL) in humans. To understand the molecular networks driving leukemia in the Ebf1+/-Pax5+/- (dHet) mouse model for B-ALL, we interrogated the transcriptional profiles and chromatin status of leukemic cells, preleukemic dHet pro-B, and wild-type pro-B cells with the corresponding EBF1 and Pax5 cistromes. In dHet B-ALL cells, many EBF1 and Pax5 target genes encoding pre-BCR signaling components and transcription factors were down-regulated, whereas Myc and genes downstream from IL-7 signaling or associated with the folate pathway were up-regulated. We show that blockade of IL-7 signaling in vivo and methotrexate treatment of leukemic cells in vitro attenuate the expansion of leukemic cells. Single-cell RNA-sequencing revealed heterogeneity of leukemic cells and identified a subset of wild-type pro-B cells with reduced Ebf1 and enhanced Myc expression that show hallmarks of dHet B-ALL cells. Thus, EBF1 and Pax5 may safeguard early stage B cells from transformation to B-ALL by limiting IL-7 signaling, folate metabolism and Myc expression.

Relapse-Fated Latent Diagnosis Subclones in Acute B Lineage Leukemia Are Drug Tolerant and Possess Distinct Metabolic Programs.

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. SIGNIFICANCE: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse.

The Biology of B-Progenitor Acute Lymphoblastic Leukemia.

Genomic analyses have revolutionized our understanding of the biology of B-progenitor acute lymphoblastic leukemia (ALL). Studies of thousands of cases across the age spectrum have revised the taxonomy of B-ALL by identifying multiple new subgroups with diverse sequence and structural initiating events that vary substantially by age at diagnosis and prognostic significance. There is a growing appreciation of the role of inherited genetic variation in predisposition to ALL and drug responsiveness and of the nature of genetic variegation and clonal evolution that may be targeted for improved diagnostic, risk stratification, disease monitoring, and therapeutic intervention. This review provides an overview of the current state of knowledge of the genetic basis of B-ALL, with an emphasis on recent discoveries that have changed our approach to diagnosis and monitoring.

Germline deletion of ETV6 in familial acute lymphoblastic leukemia

AbstractRecent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.

PAX5-driven subtypes of B-progenitor acute lymphoblastic leukemia.

Recent genomic studies have identified chromosomal rearrangements defining new subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL), however many cases lack a known initiating genetic alteration. Using integrated genomic analysis of 1,988 childhood and adult cases, we describe a revised taxonomy of B-ALL incorporating 23 subtypes defined by chromosomal rearrangements, sequence mutations or heterogeneous genomic alterations, many of which show marked variation in prevalence according to age. Two subtypes have frequent alterations of the B lymphoid transcription-factor gene PAX5. One, PAX5alt (7.4%), has diverse PAX5 alterations (rearrangements, intragenic amplifications or mutations); a second subtype is defined by PAX5 p.Pro80Arg and biallelic PAX5 alterations. We show that p.Pro80Arg impairs B lymphoid development and promotes the development of B-ALL with biallelic Pax5 alteration in vivo. These results demonstrate the utility of transcriptome sequencing to classify B-ALL and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis.

Characterization of PAX5-driven Subtypes in B-progenitor Acute Lymphoblastic Leukemia

Characterization of PAX5-driven Subtypes in B-progenitor Acute Lymphoblastic Leukemia

Germline Genetic IKZF1 Variation and Predisposition to Childhood Acute Lymphoblastic Leukemia

Somatic genetic alterations of IKZF1, which encodes the lymphoid transcription factor IKAROS, are common in high-risk B-progenitor acute lymphoblastic leukemia (ALL) and are associated with poor prognosis. Such alterations result in the acquisition of stem cell-like features, overexpression of adhesion molecules causing aberrant cell-cell and cell-stroma interaction, and decreased sensitivity to tyrosine kinase inhibitors. Here we report coding germline IKZF1 variation in familial childhood ALL and 0.9% of presumed sporadic B-ALL, identifying 28 unique variants in 45 children. The majority of variants adversely affected IKZF1 function and drug responsiveness of leukemic cells. These results identify IKZF1 as a leukemia predisposition gene, and emphasize the importance of germline genetic variation in the development of both familial and sporadic ALL.

Germline Genetic IKZF1 Variation and Predisposition to Childhood Acute Lymphoblastic Leukemia

IKZF1, which encodes the lymphoid transcription factor IKAROS, is frequently targeted by somatic deletions and mutations in high-risk Bprogenitor acute lymphoblastic leukemia (ALL) and is associated with poor prognosis. Somatic IKZF1 alterations result in the acquisition of stem celllike features, overexpression of adhesion molecules causing aberrant cellcell and cell-stroma interaction, and decreased sensitivity to tyrosine kinase inhibitors. Here we report coding IKZF1 variation in familial childhood ALL and 1% of presumed sporadic B-ALL, with 28 unique germline IKZF1 variants in 45 children. The majority of variants adversely affected IKZF1 function and drug responsiveness of leukemic cells. These results identify IKZF1 as a leukemia predisposition gene, and emphasize the importance of germline genetic variation in the development of both familial and sporadic ALL.

Conserved IKAROS-regulated genes associated with B-progenitor acute lymphoblastic leukemia outcome.

Genetic alterations disrupting the transcription factor IKZF1 (encoding IKAROS) are associated with poor outcome in B lineage acute lymphoblastic leukemia (B-ALL) and occur in >70% of the high-risk BCR-ABL1+ (Ph+) and Ph-like disease subtypes. To examine IKAROS function in this context, we have developed novel mouse models allowing reversible RNAi-based control of Ikaros expression in established B-ALL in vivo. Notably, leukemias driven by combined BCR-ABL1 expression and Ikaros suppression rapidly regress when endogenous Ikaros is restored, causing sustained disease remission or ablation. Comparison of transcriptional profiles accompanying dynamic Ikaros perturbation in murine B-ALL in vivo with two independent human B-ALL cohorts identified nine evolutionarily conserved IKAROS-repressed genes. Notably, high expression of six of these genes is associated with inferior event-free survival in both patient cohorts. Among them are EMP1, which was recently implicated in B-ALL proliferation and prednisolone resistance, and the novel target CTNND1, encoding P120-catenin. We demonstrate that elevated Ctnnd1 expression contributes to maintenance of murine B-ALL cells with compromised Ikaros function. These results suggest that IKZF1 alterations in B-ALL leads to induction of multiple genes associated with proliferation and treatment resistance, identifying potential new therapeutic targets for high-risk disease.

Ikaros: Exploiting and targeting the hematopoietic stem cell niche inB-progenitor acute lymphoblastic leukemia.

Genetic alterations of IKZF1 encoding the lymphoid transcription factor IKAROS are a hallmark of high-risk B-progenitor acute lymphoblastic leukemia (ALL), such as BCR-ABL1-positive (Ph+) and Ph-like ALL, and are associated with poor outcome even in the era of contemporary chemotherapy incorporating tyrosine kinase inhibitors. Recent experimental mouse modeling of B-progenitor ALL has shown that IKZF1 alterations have multiple effects, including arresting differentiation, skewing lineage of leukemia from myeloid to lymphoid, and, in Ph+leukemia, conferring resistance to tyrosine kinase inhibitor (TKI) therapy without abrogating ABL1 inhibition. These effects are in part mediated by acquisition of an aberrant hematopoietic stem cell-like program accompanied by induction of cell surface expression of stem cell and adhesion molecules that mediate extravascular invasion and residence in the niche and activation of integrin signaling pathways. These effects can be exploited therapeutically using several approaches. IKZF1 alterations also result in upregulation of RXRA that encodes part of the heterodimeric retinoic acid X receptor. Rexinoids, a synthetic class of retinoids that bind specifically to retinoid "X" receptors such as bexarotene potently reverse aberrant adhesion and niche mislocalization invivo and induce differentiation and cell cycle arrest. Focal adhesion kinase inhibitors block the downstream integrin-mediated signaling, reverse adhesion, and niche mislocalization. Both agents act synergistically with TKIs to prolong survival of Ph+ALL in mouse and human xenograft model, with long-term remission induced by focal adhesion kinase inhibitors. Therefore, these findings provide important new conceptual insights into the mechanisms by which IKZF1 alterations result in drug resistance and indicate that therapeutic strategies directed against the pathways deregulated by mutation, rather than attempting to restore IKZF1 expression directly, represent promising therapeutic approaches in this disease.

Deregulation of DUX4 and ERG in acute lymphoblastic leukemia.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.

Advances in the Genetics and Therapy of Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) remains an important cause of morbidity in children and adults. In this article, we highlight advances in the genetics and therapy of three key subtypes of ALL: T-cell ALL, BCR-ABL1 (Philadelphia [Ph] chromosone-positive), and Ph-like ALL. T-ALL is an aggressive disease that accounts for about 15% and 25% of ALL among pediatric and adult cohorts, respectively, and exhibits a multistep nature of cancer initiation and progression. The integration of cytogenetics, molecular biology, and immunophenotype analyses has led to the identification of defined T-ALL subgroups, such as early T-cell precursor ALL and novel lesions with a prognostic role, for which specific inhibitors are being developed. Ph-positive ALL was historically regarded as a subtype of ALL with a poor prognosis, and allogeneic stem cell transplant was recommended for all patients who could undergo this procedure. The deep complete responses seen with combination tyrosine kinase inhibitors (TKIs) and chemotherapy in Ph-positive ALL, and the reports of long-term survival among some patients not undergoing allogeneic stem cell transplant, has raised the question of whether there is a subset of patients who could be cured without this intervention. Ph-like ALL is a subtype of B-progenitor ALL common among older children and adults and associated with a diverse range of genetic alterations that activate kinase signaling. Ph-like ALL is also associated with poor outcome, for which precision medicine trials identifying kinase alterations and testing TKI therapy are being developed.

Advances in the Genetics and Therapy of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) remains an important cause of morbidity in children and adults. In this article, we highlight advances in the genetics and therapy of three key subtypes of ALL: T-cell ALL, BCR-ABL1 (Philadelphia [Ph] chromosone-positive), and Ph-like ALL. T-ALL is an aggressive disease that accounts for about 15% and 25% of ALL among pediatric and adult cohorts, respectively, and exhibits a multistep nature of cancer initiation and progression. The integration of cytogenetics, molecular biology, and immunophenotype analyses has led to the identification of defined T-ALL subgroups, such as early T-cell precursor ALL and novel lesions with a prognostic role, for which specific inhibitors are being developed. Ph-positive ALL was historically regarded as a subtype of ALL with a poor prognosis, and allogeneic stem cell transplant was recommended for all patients who could undergo this procedure. The deep complete responses seen with combination tyrosine kinase inhibitors (TKIs) and chemotherapy in Ph-positive ALL, and the reports of long-term survival among some patients not undergoing allogeneic stem cell transplant, has raised the question of whether there is a subset of patients who could be cured without this intervention. Ph-like ALL is a subtype of B-progenitor ALL common among older children and adults and associated with a diverse range of genetic alterations that activate kinase signaling. Ph-like ALL is also associated with poor outcome, for which precision medicine trials identifying kinase alterations and testing TKI therapy are being developed.

Efficacy of Focal Adhesion Kinase Inhibition in Combination with Dasatinib in BCR-ABL1 Acute Lymphoblastic Leukemia

Introduction: BCR-ABL1+ B-progenitor acute lymphoblastic leukemia (Ph+ B-ALL) is a highly aggressive disease that is often refractory to currently available therapies. Our previous genomic profiling studies have identified loss-of-function or dominant negative mutations in IKZF1, encoding the lymphoid transcription factor Ikaros, in over 80% of Ph+ ALL. In addition, deletion of CDKN2A, which encodes the INK4A and ARF tumor suppressors, is observed in approximately half of all cases (Mullighan et al., 2008). Alterations of IKZF1 are associated with poor outcome despite the use of tyrosine kinase inhibitors (TKIs). Ikzf1 alterations, including Ikaros isoform 6 (IK6), result in the acquisition of stem cell-like features, enhanced self-renewal, expression of adhesion molecules, and transcriptional upregulation of focal adhesion kinase (FAK), resulting in increased adhesion in vitro and in vivo, and decreased sensitivity to TKIs (Churchman, Cancer Cell, in press). VS-4718 is a potent, selective, and orally bioavailable FAK inhibitor currently under evaluation in a phase 1 clinical trial in subjects with various solid tumors, however in vivo efficacy in hematological malignancies had not been evaluated. Targeting FAK with VS-4718 is an attractive approach to abrogate the adhesive phenotype of IKZF1-altered leukemic cells potentially enhancing the effects of dasatinib in the treatment of high-risk BCR-ABL1 B-ALL.Methods: We examined the efficacy and mechanisms of FAK inhibition using VS-4718 as a single agent and in combination with dasatinib in vitro and in vivo in a range of xenograft and genetically engineered mouse models of BCR-ABL1 ALL. Each model had concomitant deletion of Arf which is observed in approximately 50% of human cases.Results: A pre-clinical in vivo trial of dasatinib and VS-4718 combination therapy in a murine C57Bl/6 Arf-/- BCR-ABL1 pre-B cell model resulted in a marked increase in survival in both IK6-expressing and non-IK6 cohorts of mice, and one complete long-term remission in the IK6-expressing group. Further, we showed increased efficacy of VS-4718 and dasatinib, compared to either agent alone, against two highly aggressive human Ph+ IK6-expressing B-ALL xenografts in vivo, with decreased infiltration of leukemic cells in bone marrow and spleens demonstrating a synergistic effect of the VS-4718/dasatinib combination. In vitro cell viability was reduced with induction of apoptosis at increasing concentrations of VS-4718 as a single agent, and further potentiated the effects of dasatinib in cytotoxicity assays using human xenografted and murine leukemic cells.VS-4718 profoundly diminished the ability of BCR-ABL1-expressing cells to form cell-matrix adhesions in vitro, as evident by the reduced adherence to fibronectin monolayers and bone marrow stromal cells. VS-4718 almost completely abolished the colony-forming potential of BCR-ABL1-expressing murine pre-B cells with and without Ikzf1 alterations at drug concentrations that do not affect cell viability suggestive of a reduction in self-renewal.Calvarial imaging of mice transplanted with Ikzf1-altered BCR-ABL1 leukemic cells and treated with VS-4718 alone in vivo revealed a discernible reduction in adhesion in the intact bone marrow niche of Prrx1-Cre; LSL-tdTomato recipient mice. VS-4718 treated leukemic cells localized to Prrx1-expressing perivascular endothelial cells and exhibited round morphology in contrast to the typical spindle-like appearance of Ikzf1-altered pre-B cells adhering to the bone marrow stroma, suggesting that VS-4718 treatment abolished the aberrant leukemic cell-stromal adhesion induced by Ikaros alterations in vivo.Conclusions: Direct inhibition of FAK with VS-4718 attenuates the adhesive, stem-like properties of IKZF1-altered BCR-ABL1 leukemic cells that contribute to the poor prognosis of patients treated with currently available therapies. Targeted FAK inhibition is thus a promising avenue for improving the response of BCR-ABL1 ALL to dasatinib, particularly in refractory cases harboring IKZF1 alterations. These data support the clinical development of VS-4718 in combination with dasatinib in Ph+ B-ALL. DisclosuresShapiro:Verastem: Employment, Equity Ownership. Pachter:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding; Incyte: Consultancy, Honoraria. Off Label Use: The FAK inhibitor VS-4718 for the treatment of BCR-ABL1 acute lymphoblastic leukemia in preclinical models.

High Prevalence of Relapse in Australian Children with Ph-like Acute Lymphoblastic Leukemia Despite Risk Adapted Treatment

Introduction:While remission rates for childhood acute lymphoblastic leukemia (ALL) now exceed 80%, relapsed ALL remains the leading cause of non-traumatic death in children. Recently, a high-risk group of B-progenitor ALL patients has been identified. Such cases exhibit a gene expression profile similar to that of BCR-ABL1 positive (Ph+) ALL but are BCR-ABL1 negative, and also experience poor treatment outcomes. This subset, termed Ph-like ALL, is characterised by a range of genetic alterations that activate cytokine receptor and kinase signalling, allowing potential targeting by available tyrosine kinase inhibitors (TKI). The frequency of Ph-like ALL in the Australian community and the prognosis in the setting of the first MRD (minimal residual disease) intervention trial by the Australian and New Zealand Children's Haematology/Oncology Group (ANZCHOG ALL8) is unknown.Method:We retrospectively screened 250 unselected samples that were available from children diagnosed with B-ALL, for Ph-like ALL. The children, aged between 1 and 18 years, were enrolled on the ANZCHOG ALL8 trial and recruited from 2002-2011. The criteria for stratification to the high-risk group, based upon Berlin-Frankfurt-Munster (BFM) protocols, were BCR-ABL1 or MLL t(4;11) translocation; poor prednisolone response at day 8; failure to achieve remission by day 33 or high MRD (>5 x10-4) at day 79. MRD was measured by RQ-PCR for patient-specific immunoglobulin and T-cell receptor rearrangements. All patients received a standard BFM four drug induction chemotherapy regimen including a prednisolone pre-phase and intrathecal methotrexate. High-risk patients received a further three novel intensive blocks of chemotherapy followed by transplant in most cases. Patients were screened for Ph-like ALL using a custom Taqman Low Density Array (TLDA) based upon previous reports. Fusions were then confirmed by RT-PCR for 30 known fusions, Sanger sequencing, mRNA sequencing and/or FISH.Results:Ten percent (25/250) of children in this cohort were identified as having Ph-like ALL, with most fusions converging on kinase activating pathways (Table 1). Three Ph-like ALL patients were considered high-risk, the remaining 22 (88%) were medium risk. Five children with Ph-like ALL, that did not have a fusion identified by RT-PCR, are currently under further investigation. Furthermore, 15 of the 20 (75%) of rearrangements involved CRLF2 with 10 (66%) of these children relapsing. Strikingly, 56% (14/25) of children in the ALL8 cohort who were identified as Ph-like subsequently relapsed compared to 16% (36/225) who were not, with significantly worse event free survival (p<0.0001) (Figure 1).Conclusion:Here we demonstrate a significantly higher frequency of relapse amongst Australian children with Ph-like ALL compared to non Ph-like disease despite a MRD-adjusted intensification regimen. In this cohort, these children should be classified as high-risk due to high treatment failure rates with standard/medium risk regimens. Importantly, rapid identification of these patients may guide future intervention with targeted therapies, such as TKI, matched to the causative genetic lesion in this high-risk group. [Display omitted] [Display omitted] DisclosuresHughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mullighan:Incyte: Consultancy, Honoraria; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.