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Further information on antibodies, differentially expressed genes, clinical characteristics, survival analysis, mutations, and methylation.

Purpose:To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease.Experimental Design:Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698).Results:TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment.Conclusions:The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.

Supplementary Figures 1-4 from IL-7 Contributes to the Progression of Human T-cell Acute Lymphoblastic Leukemias

Understanding the molecular and cellular processes involved in lung epithelial regeneration may fuel the development of new therapeutic approaches for lung diseases. We combined new mouse models that allow diphtheria toxin (DTA)-mediated depletion of specific epithelial cell types and GFP-labeling of dividing cells with single-cell transcriptomics to characterize the regeneration of the distal lung. We uncovered new cell types, some of which likely represent epithelial precursors, propose goblet cells as progenitor cells, and provide evidence that adventitial fibroblasts act as supporting cells in epithelial regeneration. We also found that DTA-expressing cells can persist in the lung, express specific inflammatory factors, and resemble a previously undescribed population in the lungs of COVID-19 patients. Our study provides a comprehensive single-cell atlas of the distal lung that characterizes early transcriptional and cellular responses to defined epithelial injury, encompassing proliferation, differentiation, and cell-to-cell interactions.

Purpose: To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease. Experimental Design: Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698). Results: TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment. Conclusions: The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.

Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.

Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.

© 2014 Ferrata Storti Foundation. This is an open-access paper, Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy., This study was supported by grant PTDC/IC/83023/2007, from Fundação para a Ciência e a Tecnologia (FCT). A.M.G. and V.P. received BI fellowships from FCT. L.R.M. and A.M. received a post-doctoral and a PhD fellowship, respectively, both from FCT.

Activity of the clinical-stage CK2-specific inhibitor CX-4945 against chronic lymphocytic leukemia

Mutations and deletions of the tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) are frequently involved in the development of cancer. However, PTEN is also tightly controlled by various non-genomic mechanisms. This review focuses on those mechanisms, namely on the epigenetic silencing of PTEN, post-transcriptional regulation by non-coding RNAs and post-translational modification. We summarise their involvement in cancer in general, and place some emphasis on leukaemia, where PTEN genetic lesions are relatively uncommon and, strikingly, high levels of PTEN expression frequently associate with PTEN functional inactivation. Overall, it is apparent that rather than looking strictly for PTEN genetic lesions and PTEN expression status, the key to evaluating the real impact of PTEN as a 'quasi-insufficient' tumour suppressor must rely on the complete understanding of PTEN's 'functional dose', incorporating the multiple layers of PTEN regulation in the cell that are ultimately compromised in a given cancer.

The importance of microenvironmental factors for driving progression in leukemia has been debated. Previous evidence has pointed to interleukin-7 (IL-7), a fundamental cytokine to normal T-cell development and homeostasis, as an important determinant of the viability and proliferation of T-cell acute lymphoblastic leukemia (T-ALL) cells in vitro. In this study, we report that IL-7 is also a critical determinant of T-ALL progression. T-ALL cell lines and primary T-ALL samples initiated leukemia more slowly when engrafted to immunocompromised Rag2−/−IL2rg−/− mice lacking IL-7. This effect was not related to reduced engraftment or homing of transplanted cells to the bone marrow. Instead, IL-7 deficiency diminished expansion of leukemia cells in the bone marrow and delayed leukemia-associated death of transplanted mice. Moreover, infiltration of different organs by T-ALL cells, which characterizes patients with advanced disease, was more heterogeneous and generally less efficient in IL-7–deficient mice. Leukemia progression was associated with increased Bcl-2 expression and cell viability, reduced p27Kip1 expression, and decreased cell-cycle progression. Clinical measurements of IL-7 plasma levels and IL-7 receptor (IL-7R) expression in T-ALL patients versus healthy controls confirmed that IL-7 stimulates human leukemia cells. Our results establish that IL-7 contributes to the progression of human T-cell leukemia, and they offer preclinical validation of the concept that targeting IL-7/IL-7R signaling in the tumor microenvironment could elicit therapeutic effects in T-ALL. Cancer Res; 71(14); 4780–9. ©2011 AACR.

© Springer Science+Business Media, LLC. 2011, 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., This study was supported by the grant PIC/IC/83193/2007 from Fundação para a Ciência e a Tecnologia. LRM has an FCT-SFRH PhD fellowship.

Expression of protein kinase CK2 is frequently deregulated in cancer and mounting evidence implicates CK2 in tumorigenesis. Here, we show that CK2 is overexpressed and hyperactivated in chronic lymphocytic leukemia (CLL). Inhibition of CK2 induces apoptosis of CLL cells without significantly affecting normal B and T lymphocytes. Importantly, this effect is not reversed by coculture with OP9 stromal cells, which are otherwise capable of rescuing CLL cells from in vitro spontaneous apoptosis. CLL cell death upon CK2 inhibition is mediated by inactivation of PKC, a PI3K downstream target, and correlates with increased PTEN activity, indicating that CK2 promotes CLL cell survival at least in part via PI3K-dependent signaling. Although CK2 antagonists induce significant apoptosis of CLL cells in all patient samples analyzed, sensitivity to CK2 blockade positively correlates with the percentage of CLL cells in the peripheral blood, β2 microglobulin serum levels and clinical stage. These data suggest that subsets of patients with aggressive and advanced stage disease may especially benefit from therapeutic strategies targeting CK2 function. Overall, our study indicates that CK2 plays a critical role in CLL cell survival, laying the groundwork for the inclusion of CK2 inhibitors into future therapeutic strategies.

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

BackgroundThe unique responsiveness of Vgamma9Vdelta2 T-cells, the major gammadelta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gammadelta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gammadelta T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR)-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the Vgamma9Vdelta2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by Eubacteria and Protozoa, and examined its gammadelta T-cell activation and anti-tumor properties.Methodology/principal findingsWe have performed a comparative study between HMB-PP and the anti-CD3epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gammadelta T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in Vgamma9(+) T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gammadelta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vgamma9(+) TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gammadelta T-cell activation upon re-stimulation. This ultimately translates in potent human gammadelta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice,Conclusions/significanceThe development of efficient cancer immunotherapy strategies critically depends on our capacity to maximize anti-tumor effector T-cell responses. By characterizing the intracellular mechanisms of HMB-PP-mediated activation of the highly cytotoxic Vgamma9(+) T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials.

Interleukin-4 stimulates proliferation and growth of T-cell acute lymphoblastic leukemia cells by activating mTOR signaling

T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous disease with respect to phenotype, gene expression profile and activation of particular intracellular signaling pathways. Despite very significant improvements, current therapeutic regimens still fail to cure a portion of the patients and frequently implicate the use of aggressive protocols with long-term side effects. In this review, we focused on how deregulation of critical signaling pathways, in particular Notch, PI3K/Akt, MAPK, Jak/STAT and TGF-beta, may contribute to T-ALL. Identifying the alterations that affect intracellular pathways that regulate cell cycle and apoptosis is essential to understanding the biology of this malignancy, to define more effective markers for the correct stratification of patients into appropriate therapeutic regimens and to identify novel targets for the development of specific, less detrimental therapies for T-ALL.

Abstract 2897 Specific inhibition of signaling elements essential for chronic lymphocytic leukemia (CLL) cell survival offers great promise for the design of improved therapies against this still incurable malignancy. The serine/threonine protein kinase CK2 is frequently upregulated in cancer, and mounting evidence implicates CK2 in tumorigenesis. Here, we evaluated whether CK2 is a valid target for therapeutic intervention in CLL, by testing the efficacy of CX-4945, a potent and highly specific orally available ATP-competitive inhibitor of CK2 that is undergoing phase I clinical trials for solid tumors and multiple myeloma. We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL and CLL cells, leading to the hyperactivation of PI3K signaling pathway, and consequently promoting leukemia cell survival (Silva et al, JCI 2008; Martins et al, Blood 2010). Therefore, we first analyzed the impact of CX-4945 on PTEN phosphorylation and PI3K pathway activation. Incubation of CLL cells with 20 μM CX-4945 for 2h resulted in striking downregulation of PTEN phosphorylation, indicative of increased PTEN activity, and a concomitant decrease in the activity of PI3K downstream targets Akt and PKC, as determined by Akt (S473), PKCβ (S660) and PKCδ (T550) phosphorylation in both MO1043 and primary CLL cells collected and isolated to >90% purity from the peripheral blood of untreated patients. Importantly, we confirmed that Akt phosphorylation on the CK2 direct target site (S129) was also inhibited by CX-4945. Next, we evaluated the functional impact of the CK2 inhibitor on CLL cell viability. Primary CLL cells (n=11) were cultured with 10 and 20 μM CX-4945. Both drug concentrations exerted clear pro-apoptotic effects in all cases (P The promising results obtained in vitro prompted us to assess the impact of CX-4945 on CLL tumor development in vivo. We implanted MO1043 CLL cells subcutaneously into Swiss nude mice. At day 3, all animals presented palpable tumor masses of approximately 150 mm3, and were randomly assigned into 4 groups (n=6 per group) to receive either CX-4945 alone (75mg/kg, bid, p.o.), fludarabine alone (34mg/kg, i.p., 5 days + 2 days rest, every week), the combination of both drugs, or vehicle control. A significant delay in tumor growth was observed in all of the treatment groups when compared to the control group (P Overall, our data indicate that pharmacological inhibition of CK2 is a promising therapeutic strategy in CLL that may be of special benefit to patients with aggressive and advanced stage disease. Moreover, our studies pave the way to the development of clinical trials using CX-4945 or other CK2 antagonists to manage CLL. Disclosures: Stansfield: Cylene Pharmaceuticals Inc.: Employment. Drygin:Cylene Pharmaceuticals Inc.: Employment.

Abstract 803 Characterization of the molecular mechanisms that regulate viability of B cell chronic lymphocytic leukemia (CLL) cells may provide novel insights into the biology of this incurable 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 protein serine/threonine kinase CK2 is frequently upregulated in different cancers and mounting evidence implicates CK2 in tumorigenesis. In the present study, we evaluated whether CK2 may play a significant role in CLL. Peripheral blood mononuclear cells (PBMCs) from CLL patients (n=15) and healthy controls (n=7 ) were initially compared by Western blot densitometry analysis. CK2α, but not CK2β, was significantly upregulated in primary CLL patient samples (P=0.0003; Unpaired t-test), and CK2α expression clearly correlated with CK2 in vitro kinase activity (P value=0.0165). To test the functional significance of CK2 overexpression in CLL, we cultured PBMCs collected from leukemia patients (n=53) or purified CLL cells (n=7) in the presence of the CK2-specific small molecule inhibitors TBB and DRB. As determined by Annexin V-APC/7AAD staining and analysis of procaspase 3 cleavage, both TBB and DRB clearly promoted apoptosis of CLL cells (CD19+CD5+) in a time- and dose-dependent manner. Importantly, neither CK2 antagonist induced significant cell death in the normal T-cell population (CD19-CD3+) present in each PBMC patient sample. Consequently, the percentage of normal T-cells dramatically increased upon TBB/DRB treatment. Further, B-cells from healthy controls (n=7) were not affected by TBB or DRB, confirming the selectivity of the CK2 inhibitors towards leukemia cells. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival in vitro, it did not prevent apoptosis of CLL cells mediated by CK2 inhibition. The pro-apoptotic effect of TBB/DRB did not correlate with clinical parameters such as lymphocyte doubling time, ZAP-70 expression or IGHV mutational status. Interestingly, there was a significant association between the percentage of CLL cells in the peripheral blood and sensitivity to CK2 inhibition (e.g. 25μM TBB, P=0.0043), and treatment with 12.5μM TBB correlated with Binet stage (P=0.0043). We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL cells leading to the hyperactivation of PI3K signaling pathway (Silva et al, JCI 2008). Here, we found that primary CLL samples displayed higher P-PTEN/PTEN ratios than healthy controls (P=0.0034), which correlated with CK2 expression (P=0.0064). Furthermore, treatment with TBB/DRB abrogated PTEN phosphorylation in 2 primary leukemia samples analyzed, suggesting that CK2 negatively regulates the activity of the tumor suppressor PTEN in CLL and raising the possibility that the pro-survival effect of CK2 may be dependent, at least in part, on the activation of PI3K pathway. Overall, our study suggests that CK2 plays an important role in the biology of CLL, and lays the groundwork for the inclusion of CK2 antagonists into future therapeutic options. Disclosures: No relevant conflicts of interest to declare.