LETTERS TO THE EDITOR © F e r r a t a S t o r t i F o u n d a t i o n

Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome.

Key Points• PAX5-JAK2 is the first nuclear DNA-binding JAK2 fusion protein with kinase activity.• JAK2 inhibitors block the kinase activity of PAX5-JAK2.PAX5-JAK2 has recently been identified as a novel recurrent fusion gene in B-cell precursor acute lymphoblastic leukemia, but the function of the encoded chimeric proteinhas not yet been characterized in detail. Herein we show that the PAX5-JAK2 chimera, which consists of the DNA-binding paired domain of PAX5 and the active kinase domain of JAK2, is a nuclear protein that has the ability to bind to wild-type PAX5 target loci. Moreover, our data provide compelling evidence that PAX5-JAK2 functions as a nuclear catalytically active kinase that autophosphorylates and in turn phosphorylates and activates downstream signal transducers and activators of transcription (STATs) in an apparently noncanonical mode. The chimeric protein also enables cytokine-independent growth of Ba/F3 cells and therefore possesses transforming potential. Importantly, the kinase activity of PAX5-JAK2 can be efficiently blocked by JAK2 inhibitors, rendering it a potential target for therapeutic intervention. Together, our data show that PAX5-JAK2 simultaneously deregulates the PAX5 downstream transcriptional program and activates the Janus kinase-STAT signaling cascade and thus, by interfering with these two important pathways, may promote leukemogenesis. (Blood. 2015;125(8):1282-1291 IntroductionThe fusion protein PAX5-JAK2 has been recurrently detected in B-cell precursor acute lymphoblastic leukemia (BCP-ALL).1-4 Both fusion partner proteins play key roles in hematopoiesis, and somatic mutations in their encoding genes have been found in different hematologic neoplasms. 5-7The paired box transcription factor PAX5, a master regulator of B-cell commitment and maintenance, 6 is a frequent target of genetic alterations in BCP-ALL. 5,8 In ;2% to 3% of the cases, structural rearrangements result in the expression of PAX5 in-frame fusion genes. 1,2,4,5,[8][9][10] PAX5 fusion partners comprise a heterogeneous group of genes encoding transcription factors, structural proteins, kinases, and genes with thus far unknown functions. 1,2,8,9,[11][12][13] Regardless of the functional and structural diversity of the fusion partners, a unique feature of PAX5 fusions is the retention of the PAX5 DNA-binding domain, conferring nuclear localization and the ability to occupy PAX5 target sites. 14 Generally, it is hypothesized that PAX5 fusions act as aberrant transcription factors antagonizing wild-type PAX5 function in a dominant negative mode. 1,8,9,11,[15][16][17][18] However, in a recent study, we have shown that a subset of the PAX5 fusion proteins may have a cellular context-dependent activation potential, indicating that some PAX5 fusions may also activate target genes, thus arguing against their simplified trans-dominant negative function. 14 Janus kinase 2 (JAK2) belongs to a family of nonreceptor tyrosine kinases and is involved in signal transduction from many cytokine and growth hormone receptors ...

Translocation t(12;21), resulting in the ETV6-RUNX1 (or TEL-AML1) fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia and is considered a first hit in leukemogenesis. A targeted therapy approach is not available for children with this subtype of leukemia. To identify the molecular mechanisms underlying ETV6-RUNX1-driven leukemia, we performed gene expression profiling of healthy hematopoietic progenitors in which we ectopically expressed ETV6-RUNX1. We reveal an ETV6-RUNX1-driven transcriptional network that induces proliferation, survival and cellular homeostasis. In addition, Vps34, an important regulator of autophagy, was found to be induced by ETV6-RUNX1 and up-regulated in ETV6-RUNX1-positive leukemic patient cells. We show that induction of Vps34 was transcriptionally regulated by ETV6-RUNX1 and correlated with high levels of autophagy. Knockdown of Vps34 in ETV6-RUNX1-positive cell lines severely reduced proliferation and survival. Inhibition of autophagy by hydroxychloroquine, a well-tolerated autophagy inhibitor, reduced cell viability in both ETV6-RUNX1-positive cell lines and primary acute lymphoblastic leukemia samples, and selectively sensitized primary ETV6-RUNX1-positive leukemia samples to L asparaginase. These findings reveal a causal relationship between ETV6-RUNX1 and autophagy, and provide pre-clinical evidence for the efficacy of autophagy inhibitors in ETV6-RUNX1-driven leukemia.

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