In Chronic Lymphocytic Leukemia the JAK2/STAT3 Pathway Is Constitutively Activated and Its Inhibition Leads to CLL Cell Death Unaffected by the Protective Bone Marrow Microenvironment

The bone marrow microenvironment promotes proliferation and drug resistance in chronic lymphocytic leukemia (CLL). Although ibrutinib is active in CLL, it is rarely able to clear leukemic cells protected by bone marrow mesenchymal stromal cells (BMSCs) within the marrow niche. We investigated the modulation of JAK2/STAT3 pathway in CLL by BMSCs and its targeting with AG490 (JAK2 inhibitor) or Stattic (STAT3 inhibitor). B cells collected from controls and CLL patients, were treated with medium alone, ibrutinib, JAK/Signal Transducer and Activator of Transcription (STAT) inhibitors, or both drugs, in the presence of absence of BMSCs. JAK2/STAT3 axis was evaluated by western blotting, flow cytometry, and confocal microscopy. We demonstrated that STAT3 was phosphorylated in Tyr705 in the majority of CLL patients at basal condition, and increased following co-cultures with BMSCs or IL-6. Treatment with AG490, but not Stattic, caused STAT3 and Lyn dephosphorylation, through re-activation of SHP-1, and triggered CLL apoptosis even when leukemic cells were cultured on BMSC layers. Moreover, while BMSCs hamper ibrutinib activity, the combination of ibrutinib+JAK/STAT inhibitors increase ibrutinib-mediated leukemic cell death, bypassing the pro-survival stimuli derived from BMSCs. We herein provide evidence that JAK2/STAT3 signaling might play a key role in the regulation of CLL-BMSC interactions and its inhibition enhances ibrutinib, counteracting the bone marrow niche

HS1, a Lyn Kinase Substrate, Is Abnormally Expressed in B-Chronic Lymphocytic Leukemia and Correlates with Response to Fludarabine-Based Regimen

In B-Chronic Lymphocytic Leukemia (B-CLL) kinase Lyn is overexpressed, active, abnormally distributed, and part of a cytosolic complex involving hematopoietic lineage cell-specific protein 1 (HS1). These aberrant properties of Lyn could partially explain leukemic cells’ defective apoptosis, directly or through its substrates, for example, HS1 that has been associated to apoptosis in different cell types. To verify the hypothesis of HS1 involvement in Lyn-mediated leukemic cell survival, we investigated HS1 protein in 71 untreated B-CLL patients and 26 healthy controls. We found HS1 overexpressed in leukemic as compared to normal B lymphocytes (1.38±0.54 vs 0.86±0.29, p<0.01), and when HS1 levels were correlated to clinical parameters we found a higher expression of HS1 in poor-prognosis patients. Moreover, HS1 levels significantly decreased in ex vivo leukemic cells of patients responding to a fludarabine-containing regimen. We also observed that HS1 is partially localized in the nucleus of neoplastic B cells. All these data add new information on HS1 study, hypothesizing a pivotal role of HS1 in Lyn-mediated modulation of leukemic cells’ survival and focusing, one more time, the attention on the BCR-Lyn axis as a putative target for new therapeutic strategies in this disorder

Phosphoproteomic Analysis Reveals a Different Proteomic Profile in Pediatric Patients With T-Cell Lymphoblastic Lymphoma or T-Cell Acute Lymphoblastic Leukemia

T-cell lymphoblastic lymphoma (T-LBL) and lymphoblastic leukemia (T-ALL) arise from the transformation of precursor T-cells sharing common morphological and immunophenotypic features. Despite this, T-LBL and T-ALL show different genomic/transcriptomic profiles and whether they represent two distinct disease entities or variant manifestations of the same disease is still a matter of debate. In this work, we performed a Reverse Phase Protein Array study on T-LBL and T-ALL samples and demonstrated that they are characterized by a different phosphoproteomic profile. Indeed, T-LBLs showed the hyperactivation of FAK/ERK1/2 and AKT/mTOR pathways, whereas JAK/STAT pathway was significantly hyperphosphorylated in T-ALLs. Moreover, since the only criteria for discriminating T-LBL from T-ALL is blasts' infiltration below 25% in the bone marrow and lymphoma patients can present with a percentage of blasts close to this cut-off, a biomarker that could help distinguishing the two diseases would be of great help for the clinical diagnosis and treatment decision. Pursuing this aim, we identified a proteomic signature of six proteins whose expression/activation was able to discriminate stage IV T-LBL from T-ALL. Moreover, we demonstrated that AKT hyperphosphorylation alone was able to distinguish stage IV T-LBL from both T-ALL and stage III T-LBL. Concluding, these data demonstrate that T-ALL and T-LBL bear different phosphoproteomic profiles, further sustaining the hypothesis of the two disease as different entities and paving the way for the identification of new biomarkers able to distinguish stage IV T-LBL from T-ALL disease, so far based only on BM involvement criteria

Targeting of HSP70/HSF1 Axis Abrogates In Vitro Ibrutinib-Resistance in Chronic Lymphocytic Leukemia

none18The Btk inhibitor ibrutinib has significantly changed the management of chronic lymphocytic leukemia (CLL) patients. Despite its clinical efficacy, relapses occur, and outcomes after ibrutinib failure are poor. Although BTK and PLCγ2 mutations have been found to be associated with ibrutinib resistance in a fair percentage of CLL patients, no information on resistance mechanisms is available in patients lacking these mutations. The heat shock protein of 70 kDa (HSP70) and its transcription factor heat shock factor 1 (HSF1) play a role in mediating the survival and progression of CLL, as well as taking part in drug resistance in various cancers. We demonstrated that resveratrol and related phenols were able to induce apoptosis in vitro in leukemic cells from CLL untreated patients by acting on the HSP70/HSF1 axis. The same was achieved in cells recovered from 13 CLL patients failing in vivo ibrutinib treatment. HSP70 and HSF1 levels decreased following in vitro treatment, correlating to apoptosis induction. We suggest an involvement of HSP70/HSF1 axis in controlling resistance to ibrutinib in CLL cells, since their inhibition is effective in inducing in vitro apoptosis in cells from ibrutinib refractory patients. The targeting of HSP70/HSF1 axis could represent a novel rational therapeutic strategy for CLL, also for relapsing patients.restrictedFrezzato, Federica; Visentin, Andrea; Severin, Filippo; Pizzo, Serena; Ruggeri, Edoardo; Mouawad, Nayla; Martinello, Leonardo; Pagnin, Elisa; Trimarco, Valentina; Tonini, Alessia; Carraro, Samuela; Pravato, Stefano; Imbergamo, Silvia; Manni, Sabrina; Piazza, Francesco; Brunati, Anna Maria; Facco, Monica; Trentin, LivioFrezzato, Federica; Visentin, Andrea; Severin, Filippo; Pizzo, Serena; Ruggeri, Edoardo; Mouawad, Nayla; Martinello, Leonardo; Pagnin, Elisa; Trimarco, Valentina; Tonini, Alessia; Carraro, Samuela; Pravato, Stefano; Imbergamo, Silvia; Manni, Sabrina; Piazza, Francesco; Brunati, Anna Maria; Facco, Monica; Trentin, Livi

Expression of HS1 mRNA in CLL B lymphocytes.

<p>RNA extracted from normal B lymphocytes and leukemic B cells from CLL patients were analyzed for HS1 expression and normalized on GAPDH. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039902#pone-0039902-g002" target="_blank">Figure 2A</a> shows HS1 mRNA expression of 30 CLL patients and 8 normal controls. Data obtained were evaluated for their statistical significance with the Student’s <i>t</i>-test (* p<0.05 between normal controls and CLL patients, <b>A</b>) or ANOVA (** p<0.05 between normal <i>vs</i> mutated CLL <i>vs</i> unmutated CLL, <b>B</b>; normal <i>vs</i> CD38 neg CLL <i>vs</i> CD38 pos CLL, <b>C</b>; normal <i>vs</i> 13q- and normal karyotype CLL <i>vs</i> 17p- and 11q- CLL, <b>D</b>; normal <i>vs</i> still alive patients <i>vs</i> dead patients, <b>E</b>). Medians are represented by solid lines. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039902#pone-0039902-g002" target="_blank">Figure 2F</a> represents the overall survival comparison between patients (n = 8) presenting high levels of HS1 mRNA (HS1>7.99, dotted line) and patients (n = 26) presenting low levels of HS1 mRNA (HS1<7.99, solid line); 7.99 is the median of HS1 levels in normal controls.</p

Expression of HS1 protein in CLL B lymphocytes.

<p>The lysates obtained from normal B lymphocytes and leukemic B cells from CLL patients were analyzed by immunostaining with antibody against HS1. Blots were reprobed with anti-β-actin antibody as loading control. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039902#pone-0039902-g001" target="_blank">Figure 1A</a> is representative of four CLL and four healthy subjects with the respective densitometry of HS1/β-actin ratio. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039902#pone-0039902-g001" target="_blank">Figure 1B</a> shows HS1/β-actin ratio of 71 CLL patients and 26 normal controls. Data has been normalized putting equal to 1 the ratio calculated in Jurkat cell line. Data obtained were evaluated for their statistical significance with the Student’s <i>t</i>-test (* p<0.01 between normal controls and CLL patients, <b>B</b>) or ANOVA (** p<0.01 between normal <i>vs</i> mutated CLL <i>vs</i> unmutated CLL, <b>C</b>; normal <i>vs</i> CD38 neg CLL <i>vs</i> CD38 pos CLL, <b>D</b>; normal <i>vs</i> 13q- and normal karyotype CLL <i>vs</i> 17p- and 11q- CLL, <b>E</b>; normal <i>vs</i> treated CLL <i>vs</i> untreated CLL, <b>F</b>; normal <i>vs</i> still alive patients <i>vs</i> dead patients, <b>G</b>). Medians are represented by solid lines. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039902#pone-0039902-g001" target="_blank">Figure 1H</a> represents the overall survival comparison between patients (n = 16) presenting high levels of HS1 (HS1>0.93, dotted line) and patients (n = 54) presenting low levels of HS1 (HS1<0.93, solid line); the difference between curves is statistically significant (p<0.05, Kaplan Meier). 0.93 is the median of HS1 levels in normal controls.</p

Effect of <i>in vivo</i> therapy with FLU and Cy on HS1 protein.

<p>The lysates obtained from leukemic B cells (5×10⁵ for sample) from 32 patients subject to FLU-Cy therapy were analyzed by immunostaining with antibodies against HS1 and β-actin before (-) and after (+) the administration of FLU and Cy according to FLU-Cy protocol. The same cells were processed for RNA extraction, reverse transcription in cDNA and amplification of HS1 and GAPDH by Real-Time PCR. (<b>A</b>) Histograms represent HS1 percentage of variation measured by western blotting analysis. (<b>B</b>) Histograms represent HS1 percentage of variation measured by using Real-Time PCR. (<b>C</b>) Graphics reports WBC count before and after therapy in responsive (left) and unresponsive (right) patients; the reduction of WBC count underlines the responsiveness to therapy. (<b>D</b>) The western blot in the left panel is representative of 26 patients who responded to therapy; right panel is representative of 6 patients not responding to therapy. Cy, cyclophosphamide; FLU, fludarabine.</p