Acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA) is characterized by distinct presenting features and inferior prognosis compared with pediatric ALL. We performed a ...genome-wide association study (GWAS) to comprehensively identify inherited genetic variants associated with susceptibility to AYA ALL. In the discovery GWAS, we compared genotype frequency at 635 297 single nucleotide polymorphisms (SNPs) in 308 AYA ALL cases and 6,661 non-ALL controls by using a logistic regression model with genetic ancestry as a covariate. SNPs that reached P ≤ 5 × 10−8 in GWAS were tested in an independent cohort of 162 AYA ALL cases and 5,755 non-ALL controls. We identified a single genome-wide significant susceptibility locus in GATA3: rs3824662, odds ratio (OR), 1.77 (P = 2.8 × 10−10) and rs3781093, OR, 1.73 (P = 3.2 × 10−9). These findings were validated in the replication cohort. The risk allele at rs3824662 was most frequent in Philadelphia chromosome (Ph)-like ALL but also conferred susceptibility to non–Ph-like ALL in AYAs. In 1,827 non-selected ALL cases, the risk allele frequency at this SNP was positively correlated with age at diagnosis (P = 6.29 × 10−11). Our results from this first GWAS of AYA ALL susceptibility point to unique biology underlying leukemogenesis and potentially distinct disease etiology by age group.
•In this first ALL GWAS in AYAs, we determined that inherited GATA3 variants strongly influence ALL susceptibility in this age group.•These findings revealed similarities and differences in the genetic basis of ALL susceptibility between young children and AYAs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Introduction: Despite risk stratification according to presenting clinical and genetic features, 10-25% of children with acute lymphoblastic leukemia (ALL) relapse, which is associated with a poor ...prognosis. Here, we sought to provide a comprehensive overview of the genetic alterations associated with relapse in ALL.
Methods: We studied 93 children (27 female, 43 male) diagnosed with ALL (62 B-progenitor, 25 T-lineage) between 1987 and 2008 and treated on total therapy studies XI-XVI who experienced relapse and/or a second tumor. Age at diagnosis ranged from 3 months to 18 years. Median time to relapse was 3 years (range 3 months to 10 years). Seventy patients had a single relapse, 15 cases had 2 relapses, and 8 cases developed a second tumor of different lineage (B-cell lymphoma, chronic myeloid leukemia (n=1 each) and acute myeloid leukemia (n=6)). Diagnosis, relapse and matched normal samples (n=299) were studied using Affymetrix SNP 6.0 microarrays and whole genome or whole exome sequencing.
Results: We found 2692 copy number aberrations (CNAs) with a median of 9 (range 0-109) in the diagnosis samples (n=91) compared to a median of 10 (range 0-112) in the relapse samples (n=89) and 12 (range 0-70) in subsequent samples (n=20). The number of CNAs did not differ significantly between diagnosis, relapse or subsequent samples.
We identified a 7286 non-silent single nucleotide variants (SNVs) and small insertions or deletions (indels) in 5002 genes, 1392 of which were recurrent. The median number of variants was 12 (range 0-70) at diagnosis (n=91), 21 (range 0-858) at relapse (n=91; P=0.0029 v. diagnosis) and 60 (range 10-650) in subsequent samples (n=20; P<0.0001 v. diagnosis). A subset of samples revealed very high (variant range 658-1703; 7 cases, 9 samples: all relapse or subsequent samples), or high mutation burden (variant range 104-290; 12 cases, 16 samples: 2 diagnosis, 10 relapse, and 4 subsequent samples). Genes encoding mediators of DNA repair were affected in all cases with very high mutation burden, compared to 7 of the high burden cases and 18 out of 72 other cases (most commonly genes TP53, MSH2, and MUTYH; P<0.0001).
The most frequently mutated genes were NOTCH1 (n=33), NRAS (n=24), CREBBP (n=20) and KRAS (n=16). Of the recurrently altered genes, only 87 genes were known to be affected in cancer (Cancer Gene Census, COSMIC database), of which 59 were affected in leukemia and lymphoma tissues, indicating that we have identified 1306 novel recurrently affected genes, most commonly C13orf40 and MKI67. Mutations in epigenetic regulators were particularly frequent, with genes mutated in at least 3 cases altered in over 60% of the cohort (e.g. CREBBP, EP300, MLL2, MLL3, KDM6A/B, CTCF, SETD2, TET2/3, and EZH2).
Clonal evolution analyses showed multiple patterns of evolution, with relapses sharing either few or many variants with the diagnosis sample in a frequency that reflects both predominant clones and minor subclones propagating relapse. Variants in NOTCH1, NRAS, and CREBBP were preserved from a major clone at diagnosis in 4, 6, and 5 cases respectively, but acquired at relapse or grown out from a minor subclone at diagnosis in 3, 5, and 8 cases respectively. In contrast, variants in USH2A (n=4), FOXA1 (n=3), and purine/pyrimidine synthesis pathway genes NT5C2 (n=3), PRPS1 (n=3) and NT5C1B (n=1) were exclusively found in relapse samples. Notably, the NT5C2 mutations, which are thought to confer resistance to thiopurines, were subclonal at relapse in the majority of cases.
We identified 13 cases (10 B-lineage, 3 T-lineage) in which the diagnosis and relapse were fully discordant for all CNAs and sequence mutations, only 4 of which showed a prolonged remission time (>5 years). This suggests that these patients developed a second primary malignancy and may be predisposed to leukemia development. Indeed, one case revealed focal amplifications on chromosome 1q21.1 encompassing the neuroblastoma breakpoint family genes, which are implicated in cancer development. Comprehensive germline analyses are underway.
Conclusion: This study has provided detailed insight into the genetic basis of relapse, implicating multiple new genes and pathways involved in treatment resistance, demonstrating multiple patterns of clonal evolution, and revealing an unexpectedly high frequency of genetically discordant second malignancy in relapse in ALL.
Evans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype characterized by kinase-activating alterations that are amenable to treatment with ...tyrosine kinase inhibitors. The prevalence of Ph-like ALL increases with age and accounts for over 25% of patients with B-progenitor ALL between the ages of 21-39 years. However, the frequency, outcome and genetic basis of Ph-like ALL in adults over the age of 39 is unknown. The goals of this study were to define the prevalence of Ph-like ALL across the adult age spectrum, assess response to conventional chemotherapy, and define the genetic landscape of Ph-like ALL in adults.
Methods: We studied 692 adults with B-ALL obtained from multiple groups including the Alliance (Cancer and Leukemia Group B), ECOG-ACRIN, MD Anderson Cancer Center, Northern Italy Leukemia Group, Princess Margaret Cancer Centre, SWOG and UK NCRI. The cohort was divided into three age groups: 21-39 years (median age 28±6 years, n=333), 40-59 years (median age 47±6 years, n=246) and 60-79 years (median age 67±7 years, n=101). RNA samples were screened using a Taqman low density array (LDA) card that identifies patients with the Ph-like ALL gene signature, in addition to BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, MLL-rearranged and ERG altered ALL. Cytogenetic data was also available for the majority of cases. High expression of CRLF2 was determined by the LDA card, and CRLF2 rearrangement (IGH-CRLF2 or P2RY8-CFRLF2) was confirmed using fluorescence in situ hybridization. Total stranded transcriptome sequencing (RNA-seq) using the Illumina platform was performed on 99 cases and sequencing data was analyzed using FusionCatcher and CICERO.
Results: The overall prevalence of ETV6-RUNX1, TCF3-PBX1 and ERG ALL in adults was low (1.3%, 3.6% and 3.1%, respectively), whilst the prevalence of patients with BCR-ABL1, Ph-like and MLL-rearranged ALL was 20%, 24% and 14%, respectively. Ph-like ALL comprised 26% of patients between 21-39 years of age and 20% of patients aged 40-79. Patients with BCR-ABL1 and Ph-like ALL presented with higher white blood counts at diagnosis compared to non Ph-like ALL patients (57.7 and 65.0 vs 28.5 x 109/L). Patients with Ph-like ALL were also more likely to be male compared to patients with BCR-ABL1 and non Ph-like ALL, with 66% vs 50% and 50%, respectively(p<0.0001; Fisher's exact test). The outcome of patients with Ph-like ALL was markedly inferior to other ALL subtypes (excluding patients with BCR-ABL1 and MLL rearrangement), with 5-year event free survival rates of 23.2±5.4 vs 51.2±4.7 (p<0.0001) and overall survival rates of 26.5±5.5 vs 56.3±4.6 (p<0.0001).
We then characterized the kinase-activating alterations in adult Ph-like ALL. Similar to previous reports, 99 of 186 (53%) of patients with Ph-like ALL had high expression of CRLF2. Of 75 cases tested, 56 harbored IGH-CRLF2 and 19 P2RY8-CRLF2. Of the 87 Ph-like ALL patients with low CRLF2 expression, we identified rearrangements involving tyrosine kinase or cytokine receptor genes in 45 patients: ABL1 (n=5 patients), ABL2 (n=7), CSF1R (n=1), EPOR (n=8), JAK2 (n=18), PDGFRA (n=1), PDGFRB (n=2), PTK2B (n=1) and TYK2 (n=2). Nine of these 45 fusions have not previously been identified in Ph-like ALL including MEF2D-CSF1R, HMBOX1-JAK2, SMU1-JAK2, SNX29-JAK2 (n=2 patients), ZNF340-JAK2, FIP1L1-PDGFRA, TMEM2-PTK2B and ZNF340-TYK2. Exome sequencing is being performed on cases that do not harbor a kinase fusion by RNA-seq analysis.
Conclusion: Ph-like ALL is common across the age spectrum of adult ALL, comprising over 20% of patients from ages 21-79 years, with a notably high prevalence of fusions involving JAK2. These findings warrant the development of clinical trials that assess the efficacy of tyrosine kinase inhibitors to improve the treatment outcome, similar to those that are being established for pediatric ALL.
Fielding:Amgen: Consultancy, Honoraria. Rowe:Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy. Stock:Gilead: Membership on an entity's Board of Directors or advisory committees. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Mullighan:Amgen: Honoraria; Incyte: Consultancy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Introduction: While cure rates exceed 80%, many children with B-ALL still relapse. Many of these patients (pts) display a Ph-like gene expression profile (GEP), but lack canonical BCR-ABL1 ...fusion. We have identified alternate kinase fusions in Ph-like ALL that induce cell proliferation sensitive to tyrosine kinase inhibitors (TKI) (Roberts, NEJM 2014). We report retrospective analyses of 1390 B-ALL pts, 885 NCI high risk and 505 standard risk B-ALL pts with elevated minimal residual disease.
Methods: Cases were screened using an 8-gene Taqman low-density array (LDA) PCR assay to identify the Ph-like GEP (Harvey, ASH 2013). Ph-like cases with elevated CRLF2 expression were tested for CRLF2 rearrangement (CRLF2-R; P2RY8-CRLF2 by Taqman PCR on the LDA card and IGH-CRLF2 by FISH). JAK mutations in CRLF2-R cases were tested by Sanger sequencing. Ph-like cases without CRLF2-R were tested for previously identified kinase fusions involving ABL1, ABL2, CSF1R, JAK2, NTRK3, and PDGFRB by RT-PCR. Ph-like cases without detected fusions underwent RNA-sequencing, either using standard Illumina library preparation or a customized kinome capture kit (Agilent).
Results: 339 (24%) pts were Ph-like. BCR-ABL1 (N = 45) and ETV6-RUNX1 (N = 11) were excluded from further analyses, as the former already receives TKI therapy and ETV6-RUNX1 ALL lacks targetable kinase fusions (unpublished). Of the remaining 283 Ph-like cases, 153 were CRLF2high (defined by CRLF2 expression levels on the LDA card). 61 (40%) had P2RY8-CRLF2 fusion, and of the remaining 91 CRLF2high cases, 56 of 69 tested had CRLF2-R (55 to IGH, 1 to an unknown partner). Thus, 117/130 (90%) CRLF2high Ph-like cases had a documented CRLF2 genomic lesion and 52 (44%) of these had a JAK mutation. Of the 130 Ph-like CRLF2low cases, 61 (47%) had a previously reported targetable TK fusion identified by RT-PCR, kinome capture or RNA sequencing. These included: 38 ABL class fusions (17 ABL1, 5 ABL2, 3 CSF1R, 13 PDGFRB) sensitive to imatinib/dasatinib; 14 JAK2 and 8 EPOR fusions sensitive to ruxolitinib; and 1 NTRK3 fusion sensitive to crizotinib. Nine cases had known fusions with new alternate breakpoints, and an additional 9 cases had fusions of novel N-terminal partners with known actionable C-terminal kinase genes. RNA sequencing identified 8 cases with IGH-EPOR fusions not previously captured by the kinome assay, indicating the cryptic and complex nature of this rearrangement.
Conclusion: Almost half of Ph-like pediatric B-ALL pts lacking CRLF2-R harbor altered TKs with compelling pre-clinical data that they are likely amenable to targeted therapy using FDA-approved TKIs. The COG will start real-time screening with this algorithm in 2015 and allocate pts with ABL class fusions to treatment with chemotherapy plus dasatinib.
Citation Format: Shalini C. Reshmi, Richard C. Harvey, Amy Smith, I-Ming Chen, Marc Valentine, Yu Liu, Yongjin Li, Jinghui Zhang, Kathryn G. Roberts, Ying Shao, John Easton, Debbie Payne-Turner, Meenakshi Devidas, Nyla Heerema, Andrew J. Carroll, Elizabeth A. Raetz, Michael J. Borowitz, Brent L. Wood, Anne L. Angiolillo, Michael M. Burke, Wanda L. Salzer, Patrick A. Zweidler-McKay, Karen R. Rabin, William L. Carroll, Mignon L. Loh, Stephen P. Hunger, Charles G. Mullighan, Cheryl L. Willman, Julie M. Gastier-Foster. Frequency of actionable gene fusions in patients with Philadelphia chromosome-like (Ph-like) B-acute lymphoblastic leukemia (ALL): A retrospective study from the Children's Oncology Group (COG). 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 4729. doi:10.1158/1538-7445.AM2015-4729
Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, comprising multiple subtypes characterized by aneuploidy, recurring submicroscopic gains and losses of DNA, and inter- and ...intra-chromosomal rearrangements. A common feature in ALL with 44-45 chromosomes (from here on referred to as near-diploid ALL) is the presence of a dicentric chromosome, which may lead to the formation of gene fusions and the expression of chimeric proteins. At present, however, we have a limited understanding of the nature of these chimeric proteins, but some are known or predicted to dysregulate the function of oncoproteins or hematopoietic growth factors.
We recently performed genome-wide profiling of over 120 pediatric cases with hypodiploid ALL, including 68 near-haploid cases (harboring 24-31 chromosomes), 34 low-hypodiploid cases (32-39 chromosomes) and 22 near-diploid cases, 20 of which harbored a dicentric chromosome. Near-haploid ALL is characterized by a high frequency of activating mutations of Ras signaling (mainly NF1 alterations) and IKZF3 deletions, and low-hypodiploid ALL by IKZF2, TP53 and RB1alterations. In contrast, these alterations are infrequent in dicentric ALL. To gain insight into the genetic basis of near-diploid ALL with dicentric chromosomes, we examined the 20 cases including 6 with dic(9;20), 3 with dic(7;12) and 2 with dic(9;17), while the remaining 9 cases had dicentric chromosomes involving various chromosomal arms. Affymetrix SNP 6.0 microarrays, gene expression profiling and candidate gene resequencing for 19 genes were performed for all cases, and transcriptome sequencing (mRNA Seq) on tumor and whole exome sequencing on tumor and matched normal genomic DNA have to date been carried out for 10 cases.
The most common lesion was focal deletion of CDKN2A/CDKN2B, encoding the tumor suppressors INK4/ARF (77%). Approximately one third of near-diploid cases harbored mutations targeting Ras and receptor tyrosine kinase signaling, including mutations in NRAS (18%), KRAS (9%), PTPN11 (9%) and NF1 (5%). The B-lymphoid transcription factor PAX5 was altered by deletion, sequence mutation or amplification in 59% of near-diploid ALL. PAX5 has previously been reported to be involved in gene fusions created by dicentric chromosomes in ALL, including PAX5-ETV6 in some cases with dic(9;12)(p13;p13). PAX5 has previously also been shown to be targeted by dic(9;20)(p11-13;q11), which creates several non-productive fusion transcripts.
The burden of non-synonymous single nucleotide variations (SNVs) and insertion/deletion (indel) mutations in coding- and splice regions in mRNA- and exome sequenced dicentric ALL varied with 8-25 SNVs and 4-15 indels per case. Except for the recurrent lesions stated above, the only recurrently sequence mutated gene identified in these 10 cases was NOTCH1, with mutations found in two cases, both of T-lineage. Singleton mutations were identified in e.g. IL7R, JAK3, STAT3, MYCN, PTEN and DOT1L. Fusion detection in mRNA Seq data identified 1-4 head-to-tail gene fusions per case, including the previously characterized fusions DDX3X-MLLT10, PICALM-MLLT10, MLL-AFF1 and P2RY8-CRLF2, none of which are involved in the dicentric chromosomes. The P2RY8-CRLF2 fusion was by RT-PCR shown to be present in two additional dicentric ALL cases. Further, novel in-frame fusions including ETV6-AMPH and PAX5-FBRSL1 created by dic(7;12)(p11.2;p11.2) and dic(9;12), respectively, were identified, the function of which in tumorigenesis is still unknown.
An extended mRNA Seq study is currently ongoing, investigating an additional 30 near-diploid ALL cases with a focus on the dicentric chromosomes dic(7;9), dic(9;12) and dic(9;20). Further, recurrence screening will be performed on a cohort including another 45 cases, and functional evaluation is on an ongoing basis carried out on identified fusions.
Altogether, this study is anticipated to provide critical new insights into the genetic basis of near-diploid ALL harboring a dicentric chromosome, and to shed more light on the putative chimeric genes produced by the formation of dicentric chromosomes. Further, we hope to identify new targets for therapy.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 540
Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an ...expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified common deletions of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. The IKAROS alterations commonly involve coding exons 3–6, resulting in expression of a dominant negative IKAROS isoform, IK6. Moreover, the presence of IKZF1 alterations is associated with poor outcome in BCR-ABL1 ALL. We have previously shown in a retroviral bone marrow transplant model of BCR-ABL1 ALL that Ikzf1 loss results in increased penetrance of leukemia, but the role of IK6 in the pathogenesis of ALL has not been studied. Here, we have examined the effect of the expression of Ik6 in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from C57BL/6 Arf null mice was transduced with MSCV retrovirus coexpressing p185 BCR-ABL1 and luciferase, plated for 8 days to derive pre-B cells, then transduced with MSCV retrovirus expressing either wildtype Ikaros (Ik1-RFP), Ik6-RFP, or empty vector. Expression of Ik1 was not tolerated and resulted in cell death and apoptosis. IK6 expression led to increased proliferation of p185+Arf null cells with reduced sensitivity to the BCR-ABL1 kinase inhibitor dasatinib compared to cells transduced with empty vector. Intracellular phosphosignaling analysis of Crkl phosphorylation demonstrated that this reduced sensitivity to dasatinib was independent of ABL1 inhibition. Gene expression profiling of p185+Arf null-Ik6 cells revealed a gene expression signature similar to that of human BCR-ABL1+ ALL with enrichment of hematopoietic stem cells genes as well as genes involved in B-cell receptor, Notch, and Jak-Stat signaling pathways. To test the role of Ik6 in leukemogenesis and treatment responsiveness in vivo, p185 BCR-ABL1-luciferase Arf null cells were transduced with MSCV retrovirus expressing GFP alone, Ik1-GFP, or Ik6-GFP then transplanted into lethally irradiated C57BL/6 recipients. Expression of Ik6 in vivo led to accelerated tumorigenesis and decreased survival with tumors uniformly of pre-B immunophenotype. Moreover, mice transplanted with Ik6-expressing marrow were less sensitive to dasatinib therapy (10mg/kg QD initiated 7 days post-BMT) compared to control mice (19d vs. 31.5d, p<0.001), suggesting that expression of the dominant-negative Ikaros isoform Ik6 may play a key role in resistance to therapy and poor outcome in human BCR-ABL1 positive ALL. These results indicate that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 positive ALL, and that expression of dominant negative IKZF1 isoforms influences tumor responsiveness.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Abstract 540
Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), ...an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified common deletions of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. The IKAROS alterations commonly involve coding exons 3–6, resulting in expression of a dominant negative IKAROS isoform, IK6. Moreover, the presence of IKZF1 alterations is associated with poor outcome in BCR-ABL1 ALL. We have previously shown in a retroviral bone marrow transplant model of BCR-ABL1 ALL that Ikzf1 loss results in increased penetrance of leukemia, but the role of IK6 in the pathogenesis of ALL has not been studied. Here, we have examined the effect of the expression of Ik6 in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from C57BL/6 Arf null mice was transduced with MSCV retrovirus coexpressing p185 BCR-ABL1 and luciferase, plated for 8 days to derive pre-B cells, then transduced with MSCV retrovirus expressing either wildtype Ikaros (Ik1-RFP), Ik6-RFP, or empty vector. Expression of Ik1 was not tolerated and resulted in cell death and apoptosis. IK6 expression led to increased proliferation of p185+Arf null cells with reduced sensitivity to the BCR-ABL1 kinase inhibitor dasatinib compared to cells transduced with empty vector. Intracellular phosphosignaling analysis of Crkl phosphorylation demonstrated that this reduced sensitivity to dasatinib was independent of ABL1 inhibition. Gene expression profiling of p185+Arf null-Ik6 cells revealed a gene expression signature similar to that of human BCR-ABL1+ ALL with enrichment of hematopoietic stem cells genes as well as genes involved in B-cell receptor, Notch, and Jak-Stat signaling pathways. To test the role of Ik6 in leukemogenesis and treatment responsiveness in vivo, p185 BCR-ABL1-luciferase Arf null cells were transduced with MSCV retrovirus expressing GFP alone, Ik1-GFP, or Ik6-GFP then transplanted into lethally irradiated C57BL/6 recipients. Expression of Ik6 in vivo led to accelerated tumorigenesis and decreased survival with tumors uniformly of pre-B immunophenotype. Moreover, mice transplanted with Ik6-expressing marrow were less sensitive to dasatinib therapy (10mg/kg QD initiated 7 days post-BMT) compared to control mice (19d vs. 31.5d, p<0.001), suggesting that expression of the dominant-negative Ikaros isoform Ik6 may play a key role in resistance to therapy and poor outcome in human BCR-ABL1 positive ALL. These results indicate that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 positive ALL, and that expression of dominant negative IKZF1 isoforms influences tumor responsiveness.
Disclosures:
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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Introduction: BCR-ABL1-like, or Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), is characterized by a gene expression profile similar to BCR-ABL1-positive ALL, with a broad range of ...genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We previously reported a rearrangement of EPOR, encoding the erythropoietin receptor, into the immunoglobulin heavy chain locus (IGH). The aims of this study were to define the frequency and genomic architecture of EPOR rearrangements in B-ALL and to examine their role in kinase signaling and lymphoid transformation.
Methods: Whole genome and/or transcriptome sequencing was performed on 154 Ph-like ALL cases. Sanger sequencing and fluorescent in situ hybridization were used to confirm and map the EPOR rearrangements. Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of erythropoietin (EPO). EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Epor-/- fetal liver cells were transduced with empty vector, EPOR wild-type or rearranged alleles and used for erythroid colony forming unit (CFU-E) and erythroid burst-forming unit (BFU-E) assays. Luciferase-marked xenografts of human EPOR-rearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo.
Results: Eight cases (5.2% of Ph-like ALL) harbored rearrangements of EPOR into either the IGH or immunoglobulin kappa light chain (IGK) loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR. Such rearrangements resulted in overexpression of a C-terminal truncated receptor that retained the phosphorylation site required for STAT5 activation, but lacked multiple intracytoplasmic tyrosine residues whose phosphorylation is required for normal negative regulation of the receptor. Notably, the locations of the truncation sites overlap with those arising from inherited mutations in primary familial congenital polycythemia, in which frameshift and nonsense mutations truncate the receptor. A real-time quantitative PCR assay was established to provide a diagnostic tool and to confirm that primary leukemia cells with these EPOR rearrangements overexpress N-terminal exons but lack expression of C-terminal truncated exon eight. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/- mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Expression of wild-type or truncated EPOR in Epor-/- fetal liver cells promoted erythroid differentiation with formation of CFU-E and BFU-E colonies, indicating that truncated receptors sustain erythroid development. Xenografted EPOR-rearranged leukemic cells exhibited high levels of mutant EPOR on the cell surface, constitutive STAT5 phosphorylation and sensitivity to the JAK2 inhibitor ruxolitinib ex vivo and in vivo.
Conclusions: We have identified a subset of Ph-like ALL cases characterized by rearrangements of truncated EPOR into the IGH/IGK chain loci. This represents an entirely new mechanism of EPOR deregulation and unexpectedly implicates EPOR signaling as an important factor influencing B-lymphoid malignancies that are amenable to JAK-STAT5 inhibition. Clinical trials testing ruxolitinib in ALL patients with EPOR rearrangements are warranted.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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