Effect of Different Methods of Trypsinization on Cell Viability and Clinical Outcome in Vitiligo Patients Undergoing Noncultured Epidermal Cellular Suspension

Abstract The L265P somatic mutation in the Myeloid Differentiation Primary Response 88 (MYD88) gene is recurrently observed in CLL; although this mutation has been demonstrated to have functional effects in multiple hematologic malignancies, its role in CLL is largely unknown. To address this gap in knowledge, we examined the clinical and biological impact of MYD88 L265P mutations in CLL by analysis of gene expression, cell viability and Toll-like Receptor 9 (TLR9)-induced signaling and cytokine production. Out of 160 CLL patient samples subjected to whole-exome sequencing and previously reported by our group, 10 were found to harbor MYD88 L265P mutations, all of which possessed mutated immunoglobulin heavy chain variable (IGHV) regions (p = 0.006). While IGHV mutated patients are generally known to exhibit better prognosis compared to IGHV unmutated patients, the presence of MYD88 L265P within the IGHV mutated subset was associated with earlier age of disease onset (p = 0.04) and worse overall survival (OS; p = 0.00017), comparable to IGHV unmutated samples with wild-type (WT) MYD88. No association with the presence of chromosome 13q deletions (p = 0.26) or prior treatment at the time of sampling (p = 0.10) was observed. Gene expression microarray analysis restricted to the IGHV mutated subset (MYD88 WT: n = 76; MYD88 L265P: n=10) and conducted using a PAM-based approach demonstrated that MYD88 L265P mutation was associated with differential expression of 28 genes, whose expression was then examined across all CLL samples with available gene expression data (n = 150). Using Cox modeling, a composite gene signature score was determined for each patient, who were subsequently dichotomized based on median signature. This method was able to predict both OS and event free survival (EFS) in a univariable analysis (OS: p = 1.2E-06; EFS: p = 7.6E-13). Statistical significance was maintained when a multivariable analysis was conducted, adjusting for known CLL risk factors including age, IGHV status, ZAP70 expression, cytogenetics and prior treatment (p < 0.0001 for OS and EFS). The univariable (OS: p = 1.6E-05; EFS: p = 5.7E-10) and multivariable findings (p < 0.003 for OS and EFS) were further confirmed in an independent validation cohort (n = 87). To identify a more parsimonious gene set, we applied a L1 penalized proportional hazards model to the discovery and validation cohorts, separately. This approach identified 5 overlapping genes (BCAT1, BMP6, CHAD, IKZF2, and TRIO) between the two cohorts that appear to be the main drivers of the predictive signature. To inhibit MYD88 signaling in CLL cells, we treated MYD88 L265P and WT cells (n = 6/group, matched for clinical characteristics: IGHV, ZAP70, cytogenetic, and treatment status) in vitro with a highly-selective small molecule IRAK4 inhibitor, ND-2158 (Nimbus Therapeutics). ND-2158 significantly reduced cell viability in a dose dependent manner in both MYD88 WT and L265P primary CLL cells, either alone or in combination with a fixed concentration of the B-cell receptor (BCR) pathway inhibitor, ibrutinib. The TLR9 agonist CpG was used to stimulate signaling through the MYD88 pathway in vitro. ND-2158 inhibition of CpG-induced IRAK4 activation in CLL cells (n = 3/group, matched for clinical characteristics) blocked IRAK1 and IκBα degradation and led to a dose-dependent decrease in the ratio of phospho/total proteins. No significant differences were noted between MYD88 WT and L265P samples, consistent with our cell viability results. CLL-secreted levels of IL-6, IL-10 and CCL3 were measured in culture supernatants treated with ND-2158+/- CpG stimulation (n = 4/group, matched for clinical characteristics). CpG stimulated cytokine levels (p < 0.0001 for all cytokines+/- CpG) were significantly inhibited in a dose-dependent manner by ND-2158. Again, no significant differences were observed between MYD88 WT and L265P CLL with respect to cytokine production, either at baseline or in CpG-stimulated DMSO treated control cells. In conclusion, the differences in clinical outcome and gene expression observed between MYD88 WT and L265P IGHV mutated CLLs indicate a functional role for MYD88 L265P in CLL. The inferior clinical outcome in IGHV mutated CLL with L265P mutation suggests that MYD88 signaling may be a relevant target in CLL. ND-2158 inhibits signaling in the MYD88 pathway, suggesting potential therapeutic utility of IRAK4 inhibitors in CLL. Disclosures Chaudhary: Nimbus Therapeutics: Equity Ownership. Miao:Nimbus Therapeutics: Employment. Westlin:Nimbus Therapeutics: Employment.

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Adaptive Reactivation of Signaling Pathways As a Novel Mechanism of Resistance to JAK Inhibitors in Ph-like ALL

Blood ◽

10.1182/blood.v128.22.755.755 ◽

2016 ◽

Vol 128 (22) ◽

pp. 755-755 ◽

Cited By ~ 1

Author(s):

Christian Hurtz ◽

Sarah K Tasian ◽

Gerald Wertheim ◽

Rachel Astles ◽

Alexis Zebrowski ◽

...

Keyword(s):

Cell Proliferation ◽

Clinical Outcome ◽

Cell Viability ◽

Older Patients ◽

Cytokine Receptor ◽

Constitutive Activation ◽

Pi3k Inhibition ◽

Apoptotic Effect ◽

Receptor Signaling Pathway ◽

Dual Treatment

Abstract Background: Ph-like ALL was first described in 2009 and was accepted as a novel B-ALL subgroup by the WHO in 2016. Ph-like ALL is characterized by different chromosomal rearrangements involving CRLF2, ABL1, JAK2, and EPOR. In addition, mutations in PAX5, IKZF1, and JAK2 are very common. The gene expression profile of this disease is very similar to Ph+ ALL which is characterized by the BCR-ABL1 oncogene. We have previously shown that Ph-like ALL is a high risk disease with a poor prognosis and constitutive activation of cytokine receptor signaling pathway (Tasian et al., submitted). Our current study is focusing on redundant signaling activation in Ph-like ALL that allows the cells to survive cytokine receptor inhibition. Results: We and others have shown that Ph-like ALL have constitutive activation of the cytokine receptor signaling pathway evident by high expression levels of pSTAT5. Xenotransplantation models of Ph-like ALL show a decrease in leukemia burden when treated with Ruxolitinib a JAK1/2 inhibitor. However, all mice ultimately succumb to the disease, demonstrating that Ph-like ALL does not demonstrate oncogene addiction. To understand these results, we have initially used Ph-like ALL cell lines. Ruxolitinib treatment of such cell lines decreases survival but does not eliminate all viable cells. In addition, Ruxolitinib leads to down regulation of pSTAT5, pSTAT3, pAKTT308 as well as pAKTS473. To further study the mechanism of resistance to JAK inhibition we performed a time course experiments and found that as early as 48-72 hours after the initial JAK inhibitor treatment Ph-like ALL cell are able to reactivate pSTAT5 and pAKT. This effect was not due to drug inactivation. Based on this observation, we hypothesized that adaptive signaling through PI3 kinase protects Ph-like ALL cells from cell death. Interestingly, PI3K inhibition (Idelalisib) alone had only a minor effect on cell viability and cell proliferation and the Ph-like ALL cells were able to reactivate the signaling network within 48-72h. Dual treatment with JAK1/2 and PI3 kinase inhibition significantly increased the apoptotic effect and stopped proliferation. In addition, the dual treatment induced a long lasting inhibitory effect with no signs of pAKT reactivation. Strikingly, combining JAK and PI3K inhibition with Methotrexate an antimetabolite that is used for the treatment of ALL patients significantly increased the apoptotic effect of the combinatory treatment. Overall, these results demonstrate that adaptive signaling with reactivation of the PI3K signaling cascade protects Ph-like ALL cells from inhibition of JAK kinases and suggests that dual kinase inhibitor therapy may increase response in the clinic. Experiments studying how reactivation occurs and the in vivo response to dual JAK-PI3K inhibition are in progress. Clinical relevance: Older patients (>40 years) often do not qualify for induction chemotherapy as they do not tolerate high doses of chemotherapy regimens and are therefore in clinical need of alternative treatment regimens that can improve their clinical outcome. While children with ALL currently have a 5-year survival rate of 90% adults especially older adults have a very poor clinical outcome with survival rates of about 40%. We therefore combined JAK1/2 and PI3K inhibitory treatments with methotrexate a frequently used antimetabolite that is well tolerated by older patients. Strikingly, the combinatory treatment had a strong effect on cell viability and represents so far the first study using specific signaling inhibitors combined with an antimetabolite treatment in Ph-like ALL. Conclusion: These findings identify that Ph-like ALL are able to reestablish their signaling networks. Based on our finding it is important to target more than one signaling pathways to fully inhibit cell proliferation and cell survival. We propose to treat Ph-like ALL patients with a combinatory treatment of JAK1/2 and PI3K inhibitor and currently used antimetabolite drugs to reduce the risk of relapse and to improve the clinical outcome of patients with Ph-like ALL. Disclosures Perl: Seattle Genetics: Consultancy; Actinium Pharmaceuticals, Inc.: Research Funding.

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ABCB1 rs1045642 C>T Change Is Associated with AML Cell Line Sensitivity to Calicheamicin

Blood ◽

10.1182/blood-2018-99-119648 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1364-1364

Author(s):

Roya Rafiee ◽

Soheil Meshinchi ◽

Jatinder K Lamba

Keyword(s):

Clinical Outcome ◽

Cell Viability ◽

Conflicts Of Interest ◽

Gemtuzumab Ozogamicin ◽

Leukemic Cells ◽

Live Cells ◽

Patient Response ◽

Improved Outcomes ◽

Dna Strand

Abstract Introduction: Gemtuzumab ozogamicin (GO), a CD33-antibody conjugated to DNA damaging cytotoxin-calicheamcin is a re-emerging and promising drug in AML treatment. Promising results from multiple clinical trials demonstrated GO improved outcomes in both adult and pediatric patients with most pronounced effect in favorable-risk cytogenetics. In light of these results GO was reapproved for treatment of AML by FDA in Sep 2017. Given the anticipated increase in GO use post approval, it is very timely to understand mechanisms contributing to differences in patient response. Calicheamicin releases intracellularly and causes DNA strand breaks which leads to apoptosis and cell death. The anti-leukemic effects of GO are thus entirely due to calicheamicin-induced DNA damage. Given that calicheamicin is a substrate of drug transporter ABCB1 (Pgp1), genetic polymorphisms in ABCB1 can impact the intra-cellular accumulation of calicheamicin and thus clinical outcome. Our group have recently shown that ABCB1 synonymous SNP rs1045642 C>T (also well known as 3435C>T) is associated with clinical outcome in children with AML trial, now we report the results of the in vitro evaluation of impact of ABCB1 SNP on calicheamicin mediated cell cytotoxicity. Methods: To understand differences in in vitro calicheamicin toxicity mediated by ABCB1 variation, HL-60 cells with low native ABCB1 expression were transfected with expression plasmid of ABCB1*1 (rs1045642-C and also WT for other two other SNPs in ABCB1) (WT Plasmid), and rs1045642-T. Cells were harvested 48 hours' post-transfection and treated with 40nM calicheamicin for 24 hours. Posttreatment cell were stained with permeant nuclear stain (NucRed® 647 Live Invitrogen) that emits bright far-red fluorescence upon binding to DNA in live cells. One-hour after incubation with the dye, calicheamicin toxicity was assessed by checking cell viability using flow cytometry (BDTM LSR II) and Nikon fluoresce microscope for additional visualization. Results: As shown in Figure 1a, our previous data show rs1045642 a synonymous coding change in ABCB1, in patients treated with GO presence of variant allele (CT/TT) is associated with significantly improved outcome as compared to patients with CC genotype (risk of relapse CC=45±10%, CT=30±7% TT=28±10%, CC vs. CT/TT p=0.007). Consistent with these results we observe significant decrease in cell viability with transfection with ABCB1-rs1045642/3435T as compared to the ABCB1-rs1045642/3435-C (P < 0.05) expression constructs. These results indicate that higher levels of calicheamicin toxicity in ABCB1-3435T transfected cells as opposed to cells transfected with the WT plasmid (Figure 1b and c) probably due to impact of T allele on ABCB1 levels. rs1045642-T allele has been shown to be associated with lower ABCB1 expression in several studies and with higher expression in one study. Hofmeyer et al has previously inked TT genotype of rs1045642 with lower expression and activity of ABCB1 which is consistent with our observation with respect to calicheamicin. Although more work is required to establish the clear relationship between ABCB1 genotypes and calicheamicin efflux, our results suggest that presence of ABCB1- low expressing rs1045642 TT genotype might be resulting in lower-efflux and thus higher intracellular abundance of calicheamicin in leukemic cells which in turn makes cells more sensitive to calicheamicin. Acknowledgments: Funding support from R21CA155524 and Children's Oncology group Disclosures No relevant conflicts of interest to declare.

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Three Different Jak2/Stat3-Related Pathways Favor the Survival of Chronic Lymphocytic Leukemia Neoplastic Clone

Blood ◽

10.1182/blood-2018-99-114591 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4405-4405

Author(s):

Filippo Severin ◽

Federica Frezzato ◽

Veronica Martini ◽

Flavia Raggi ◽

Valentina Trimarco ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Clinical Outcome ◽

Cell Viability ◽

B Lymphocytes ◽

P38 Mapk ◽

Mitochondrial Respiration ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Starting Point

Abstract INTRODUCTION Chronic lymphoproliferative disorders are characterized by the expansion of malignant lymphocytes, the most common form being Chronic Lymphocytic Leukemia (CLL). Besides intrinsic abnormalities, the acquisition of the transformed phenotype and the diffusion of disease are related to the favorable cross-talking tumor cell-microenvironment. Furthermore, it has been demonstrated that CLL cells own an amplified mitochondrial respiration leading to an increased Reactive Oxygen Species (ROS) production and intrinsic oxidative stress. Several molecules released by microenvironmental partners signal through JAK-STAT pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. We previously analyzed STAT3 and JAK2 expression, phosphorylation and localization in normal and leukemic B cells, demonstrating an abnormal activation of this pathway in the neoplastic clone with respect to normal B lymphocytes. We focused on STAT3 constitutive phosphorylation at serine (Ser) 727 residue since it has been recently observed that the presence of mt-STAT3 Ser727 promotes mitochondrial respiration and, generally, cell viability. We hypothesized the involvement of JAK2/STAT3 axis in 3 different pathways: (i) canonical "IL-6 related pathway"; (ii) JAK2/STAT3 - BCR/Lyn crosstalk; (iii) STAT3 effects on mitochondrial regulation. METHODS STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC). Purified cells (2x106 cells/ml) were cultured, and treated with the JAK2 inhibitor AG490 (10, 50 and 100μM) and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs and with/without Ibrutinib (2.5μM) and Venetoclax (1nM). CLL and normal B cell viability was tested with Annexin V/PI test by FC. P-STAT3 Ser727 expression has been correlated with p44/42, SAPK-JNK, NF-kB p65 and p38 MAPK activation by Reverse Phase Protein Microarrays (RPPA) in 57 therapy-free patients presenting good (mutated IGHV, absence of CD38 or normal karyotype) and poor (unmutated IGHV, CD38 expression or 17p deletion) prognostic factors. RESULTS We found that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. We demonstrated that STAT3 and JAK2 were similarly overexpressed in both good and poor prognosis CLL patients. However, a significant correlation between STAT3 expression levels and their overall survival was observed. Considering STAT3 over-expression and its correlation with the clinical outcome in CLL and, according to our hypothesis, we moved forward with the analysis of the three different JAK2/STAT3 pathways. We demonstrated that AG490 and Stattic were able to induce a dose-dependent apoptosis in CLL cells, also bypassing environmental protection. AG490, targeting JAK2, inhibited the phosphorylation of SHP-1 at Ser591, activating the phosphatase. In turn, SHP-1 activation led to Lyn Tyr396 dephosphorylation/inactivation. Treatment with Stattic did not affect Lyn and SHP-1 phosphorylation since this inhibitor acts downstream to AG490. In fact, simultaneous administration of Ibrutinib led to an increase of apoptosis only in Stattic, but not in AG490 treated cells. This confirms a possible dual role of JAK2 inhibition. Venetoclax/AG490 and Venetoclax/Stattic co-treatment did not show an increased cell death rate, consistent with a downstream effect of Venetoclax to both AG490 and Stattic. RPPA analysis demonstrated a correlation between STAT3, Ser727 constitutively phosphorylated in CLL, and P-p44/42 Thr202/Tyr204, P-SAPK-JNK Thr-183/Tyr185, NF-kB p65 Ser-536 and p38 MAPK Thr-180/Tyr-182 expression. All these proteins are described as involved in an alternative pathway that allows STAT3 Ser727 to exert a pro-survival effect thorough the regulation of mitochondrial activity. CONCLUSIONS The analysis of JAK2 and STAT3 expression, activation and inhibition lets us to highlight the importance of JAK2/STAT3 axis in the maintenance of 3 different survival pathways in CLL B cells. Furthermore, the correlation we demonstrated with clinical outcome of patients and the strengthening of Ibrutinib effect when we targeted this axis could represent a starting point for the development of new therapeutic strategies in CLL. Disclosures Trentin: Abbvie: Honoraria; Gilead: Research Funding; Janssen: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees.

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