Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R-mediated signaling in T-ALL.

IntroductionB-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by the accumulation of mature, monoclonal B lymphocytes in the blood, bone marrow, and secondary lymphoid organs. 1 The expansion of CLL malignant B cells appears to result mainly from a decrease in apoptosis. 2 Clinically, CLL is a very heterogeneous disease with overall survival raging from a few years to decades. Prognostic indicators include immunoglobulin heavy chain variable region (IgV H ) mutation status, cytogenetic abnormalities, clinical stage, lymphocyte doubling time, leukocyte counts, serum lactate dehydrogenase and ␤2 microglobulin levels, and ZAP-70 and CD38 expression. 3 Despite significant advances CLL is still an incurable disease, 1 and the identification of new therapeutic strategies is warranted. Characterization of the molecular mechanisms that regulate leukemia cell viability may provide novel insights into the biology of this malignancy and reveal prognostic markers and therapeutic targets. In particular, specific inhibition of signaling elements essential for leukemia cell survival offers great promise for the design of more efficient and selective therapies.The ubiquitous serine/threonine protein kinase CK2, a tetramer consisting of 2 catalytic (␣ and/or ␣Ј) and 2 regulatory ␤ subunits, is highly pleiotropic and intervenes laterally on many signaling pathways. 4,5 CK2 can drive tumorigenesis by different mechanisms, playing a global antiapoptotic role, enhancing multidrug resistance, activating the chaperone machinery that protects the oncokinome, and sustaining neo-vascularization. 6 Overexpression of CK2 has been consistently observed in human cancers, including lung, 7 kidney, 8 head and neck, 9 prostate, 10 and breast. 11 Importantly, inhibition of CK2 activity, using specific pharmacologic inhibitors or silencing the catalytic subunit, has been shown to decrease the survival of multiple myeloma, 12 acute myeloid leukemia, 13 and T-cell acute lymphoblastic leukemia 14 cells. In CLL, the genes that code for CK2␣ (CSNK2A1) and CK2␤ (CSNK2B) were identified as a part of a poor prognosis cluster associated with shorter treatment-free survival. 15 Primary CLL cells display constitutive activation of PI3K kinase activity, 16 which appears to be critical for CLL cell survival. 17,18 PTEN, the main negative regulator of PI3K signaling pathway, can be phosphorylated by CK2 at the C terminus, leading to PTEN functional inactivation and concomitant increased protein stability. 19 We recently showed that CK2 overexpression/hyperactivation in primary T-cell acute lymphoblastic leukemia cells induces PTEN nondeletional posttranslational inactivation by phosphorylation and consequent PI3K pathway hyperactivation. 14 However, the relative expression and functional impact of CK2 in CLL remains to be established.In the current study, we demonstrate that CK2 plays a critical role in the maintenance of CLL cell viability. Primary CLL cells displayed significantly higher CK2 expre...

T-cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset of ALL, the most frequent childhood malignancy. Whereas interleukin-7 (IL-7) is essential for normal T-cell development, it can also accelerate T-ALL development in vivo and leukemia cell survival and proliferation by activating phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin signaling. Here, we investigated whether STAT5 could also mediate IL-7 T-ALL-promoting effects. We show that IL-7 induces STAT pathway activation in T-ALL cells and that STAT5 inactivation prevents IL-7-mediated T-ALL cell viability, growth, and proliferation. At the molecular level, STAT5 is required for IL-7-induced downregulation of p27 and upregulation of the transferrin receptor, CD71. Surprisingly, STAT5 inhibition does not significantly affect IL-7-mediated Bcl-2 upregulation, suggesting that, contrary to normal T-cells, STAT5 promotes leukemia cell survival through a Bcl-2-independent mechanism. STAT5 chromatin immunoprecipitation sequencing and RNA sequencing reveal a diverse IL-7-driven STAT5-dependent transcriptional program in T-ALL cells, which includes inactivation by alternative transcription and upregulation of the oncogenic serine/threonine kinase Pharmacological inhibition of PIM1 abrogates IL-7-mediated proliferation on T-ALL cells, indicating that strategies involving the use of PIM kinase small-molecule inhibitors may have therapeutic potential against a majority of leukemias that rely on IL-7 receptor (IL-7R) signaling. Overall, our results demonstrate that STAT5, in part by upregulating PIM1 activity, plays a major role in mediating the leukemia-promoting effects of IL-7/IL-7R.

Specific inhibition of signaling elements essential for the viability of B-cell chronic lymphocytic leukemia (CLL) cells offers great promise for the design of more efficient therapies. The protein serine/threonine kinase CK2 is frequently upregulated in cancer, and it is overexpressed and hyperactivated in primary CLL cells from untreated patients. We have shown that inhibition of CK2 induces apoptosis of CLL cells, whereas it does not significantly impact normal lymphocytes, demonstrating the selectivity of the CK2 inhibitors toward leukemia cells. Notably, although co-culture with OP9 stromal cells and BCR stimulation both promote leukemia cell survival in vitro, they do not prevent apoptosis of CLL cells treated with CK2 inhibitors. PI3K signaling pathway was previously shown to be essential for CLL cell viability, an observation we confirmed in all patient samples analyzed. Further, we observed that CK2 blockade decreases PTEN phosphorylation, leading to PTEN activation, and that apoptosis of CLL cells upon CK2 inhibition is mediated by PKC inactivation. This suggests that activation of PI3K/PKC signaling pathway is involved in the pro-survival effects of CK2 in CLL cells. Sensitivity to CK2 inhibition does not correlate with expression of ZAP-70 or CD38, or with IGVH mutation status. However, it positively correlates with the percentage of CLL cells in the peripheral blood, β2 microglobulin levels, and Binet clinical stage. CK2 appears to play an important role in the biology of CLL and constitutes a promising target for the development of leukemia-specific therapies.

Background: T-cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset of ALL, the most frequent childhood malignancy. Although risk-adjusted chemotherapeutic regimens are currently extremely effective, their efficacy is associated with significant long-term side effects. Moreover, a significant fraction of the patients still relapse despite intensive chemotherapy, prompting the need for a deeper understanding of T-ALL biology in order to develop novel therapies. Interleukin-7 is a cytokine essential for normal T-cell development, where it has a pivotal role in promoting thymocyte survival via Bcl-2 upregulation. In this normal setting, Bcl-2 is under the control of STAT5 mediated signaling. Previously, we have shown that IL-7 promotes T-ALL expansion in vivo and leukemia cell survival and proliferation in vitro by activating PI3K/Akt/mTOR signaling pathway, consequently leading to p27kip1 downregulation and Bcl-2 upregulation. However, it is also known that T-cell lymphomas arising spontaneously in IL-7 transgenic mice depend on STAT5 activity and that leukemias displaying IL7R gain-of-function mutations are sensitive to JAK and STAT5 inhibitors. Thus, we investigated whether STAT5 could also be involved in the IL-7 pro-leukemia effects in human T-ALL cells. Methods: We used an IL-7-dependent leukemia T-cell line (TAIL7), an IL-7-responsive T-ALL cell line (HPB-ALL, with or without shRNA-mediated STAT5 silencing), primary T-ALL samples collected at diagnosis and patient-derived xenografts (PDX) and treated them with inhibitors of STAT5 (N-((4-Oxo-4H-chromen-3-yl) methylene) nicotinohydrazide) and PIM (AZD1208). Analysis of viability, cell size, cell cycle, surface CD71 and Bcl-2 expression was performed by flow cytometry. Signaling pathway activation, STAT5, PIM1, Bcl-xL, Mcl-1 and cell cycle protein expression was performed by immunoblot analysis. Proliferation was assessed by 3H-Thymidine incorporation. STAT5 ChIP-seq and RNA-seq were performed on TAIL7 cells. ChIP-PCR of histone marks H3K4me3, H3K27me, H3K27ac was performed in TAIL7 and HPB-ALL. Results: IL-7 induces JAK/STAT5 pathway activation in T-ALL cells and STAT5 genetic or pharmacological inactivation prevents IL-7-mediated T-ALL cell viability, growth and proliferation. At the molecular level, STAT5 is required for IL-7-induced downregulation of p27kip1, and upregulation of Cyclin A and TfR/CD71. Surprisingly, STAT5 inhibition does not significantly affect IL-7-mediated Bcl-2 upregulation, suggesting that, contrary to normal T-cells, STAT5 promotes leukemia cell survival via a Bcl-2-independent mechanism. In addition, IL-7-mediated increase in transcription of BCL2, BCL2L1 (Bcl-xL) and MCL1 is not affected by STAT5 silencing. To understand how STAT5 mediates the survival effects of IL-7 in T-ALL cells without affecting BCL2 transcription, we performed STAT5 ChIP-seq together with RNA-seq. Data cross-analysis reveal a diverse IL-7-driven STAT5-dependent transcriptional program in T-ALL cells, which includes transcription of the serine/threonine kinase PIM1. PIM1 is involved in cell cycle regulation and apoptosis, thereby constituting a possible alternative to Bcl-2-dependent prevention of apoptosis. We show that STAT5 silencing prevents IL-7-mediated PIM1 expression and the upregulation of active chromatin marks, H3K4me3 and H3K27ac, at the STAT5 binding region in the PIM1 gene. Notably, pharmacological inhibition of PIM kinase abrogates IL-7-mediated T-ALL cell growth and proliferation, however, without affecting cell survival. In agreement, PIM inhibition does not affect expression of Bcl-2 or Bcl-2 family anti-apoptotic members Bcl-xL and MCL1. Conclusion: Here we present evidence that T-ALL cells may have an alternative wiring of signaling networks downstream of IL-7 to that present in normal T-cells. In contrast to healthy lymphoid cells, IL-7-mediated control of survival of T-ALL cells via STAT5 does not rely on modulation of Bcl-2. Moreover, exploration of STAT5 downstream signaling reveals that PIM1 is required for IL-7-mediated proliferation of human T-ALL cells, indicating that strategies involving the use of PIM kinase small molecule inhibitors may have therapeutic potential against leukemias that rely on IL-7R and STAT5 signaling. Disclosures Barata: Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents.