ALBERT

Description: Recent studies have indicated that patients who receive stem cell transplantation (SCT) and an adjuvant rituximab demonstrate an increased risk of developing hypogammaglobulinemia. We have found that such hypogammaglobulinemia were due to the delayed recovery of memory B cells with an abnormal cell marker expression and impaired immunoglobulin production in vitro. (Nishio et al. Eur J Haemtol, 2006, Nishio et al. Br J Haematol, 2007) However, no speculation has been made regarding what factor(s) determined the risk of developing hypogammaglobulinemia after autologous SCT with the identical conditioning regimen and rituximab. Accumulated evidences have shown that FCGR3A of valine (V) allele has a higher affinity to human IgG than the phenylalanine (F) allele, and that cells bearing the FCGR3A V allele mediate antibody dependent cellular cytotoxicity more effectively. Compatibly, previous clinical studies that have examined single nucleotide polymorphisms (SNPs) of Fc receptor genes demonstrated that FCGR3A gene SNPs are associated with the response to rituximab, as a single agent, in patients with follicular lymphoma or Waldenstrom’s macroglobulinemia. These findings suggest that FCGR3A SNPs may be related to the levels of immunoglobulin after SCT and an adjuvant rituximab. To clarify this hypothesis, the FCGR3A-158V/F genotype and the levels of serum immunoglobulin six months after SCT were tested in twenty non- Hodgkin’s lymphoma (NHL) patients having received autlogous peripheral blood stem cell transplantation (APBSCT) with an adjuvant rituximab. We also compared the levels of immunoglobulin in ten NHL patients who received an identical conditioning regimen and APBSCT, but no rituximab (control group). Of the twenty patients tested for the FCGR3A-158V/F polymorphism, seven patients (35%) had homozygous F/F (158 F/F), 12 (60%) had heterozygous V/F (158 V/F), and one (5%) had homozygous V/V (158 V/V). Since only one patient was found to have 158 V/V polymorphism in this study, we defined those patients who had 158 F/F as the low affinity group, while those who had at least one 158 V allele were defined as the high affinity group following the previous definition by Anolik et al (Arthritis Rheum 2003). The three groups were not different in terms of gender, age, the disease stage, bone marrow involvement or number of extranodal sites involved at diagnosis. Before starting induction therapy, there was no significant difference in the levels of immunoglobulin among three groups. However, after APBSCT, the levels of IgG were significantly lower in the low affinity group (6.87 ± 2.38 g/l) than those in the high affinity group (10.20 ± 2.43 g/l) and control group (10.64 ± 3.04 g/l; both P

Description: Abstract 2421 Members of the nuclear factor-kB (NF-κB) family of transcription factors play important roles in cell-signaling involved in host-defense, immune responses, inflammation, and cancer. Multiple stimuli operating through different receptors are capable of activating NF-κB, including the Tumor Necrosis Factor (TNF) family cytokines, Toll-like receptors (TLRs), Nucleotide-binding Leucine Rich Repeat Proteins (NLRs), DNA-damaging agents, and others. Drugs that selectively block activation of NF-κB through one or more of these receptors could have activity against various types of cancer in which activation of NF-κB plays a role. We used a chemical biology strategy to identify novel chemical inhibitors of cell pathways responsible for NF-κB activation. Screening of a large chemical library (〉330K compounds from NIH) was conducted using human 697 pre-B-cell leukemia cells with a stably integrated NF-κB-luciferase reporter gene and stimulating the Carma/Bcl-10/MALT pathway using protein kinase C (PKC) activators (PMA/Ionomycin). Confirmed hits were counter-screened against HEK293T-NF-κB-luciferase cells treated with TNF-a, with the goal to eliminate non-pathway specific inhibitors of NF-κB. Among the hit compounds, we identified oxadiazole- and oxazole-based chemical probes as potential cell lineage- and cell differentiation-specific inhibitors of NF-κB (“NLDSi” = NF-κB lineage, differentiation-specific inhibitors). Using a panel of cell lines with stably integrated NF-κB-driven luciferase reporter genes, NLDSi compounds suppressed PMA/Ionomycin-induced NF-κB activity in a concentration-dependent manner (IC50=0.1–1.0 μM) in the pre-B-cell leukemia cell-line 697, but not in the T-cell leukemia line JURKAT, the myeloma cell-line RPMI8226, or HEK293 epithelial cells. These compounds also suppressed NF-κB activity (measured by reporter gene) in 697 cells stimulated with BAFF or treated with doxorubicin. Examining NF-κB target gene expression by q-RT-PCR (mRNA level) and immunoblotting (protein level) showed that NLDSi compounds reduced PMA/Ionomycin-induced TRAF1 and A20 expression in 697 cells and in the pre-B-cell leukemia cell line REH, but not in several mature B-cell lines, including BJAB, Daudi, OCILy3, and OCILy10. NLDSi compounds also inhibited NF-κB target gene expression in pre-B-cell lines stimulated with BAFF or treated with lipopolysaccharide (LPS) or doxorubicin (Dox). Nevertheless we found these compounds could suppress of PMA/Ionomycin-induced expression of TRAF1 and A20 gene in the primary leukemia cells of patients with chronic lymphocytic leukemia (CLL). Taken together, the compounds described here appear to selectively suppress NF-κB activation in the B-cell lineage at specific stages of differentiation. As such, these small molecules could serve as chemical probes for uncovering novel targets that regulate NF-κB activity and potentially lead to development of therapies that selectively block certain types of NF-κB activation in pre-B cell ALL, CLL, or other hematologic malignancies. (Supported by NIH grants X01 MH077633-1, U54–005033, and P01-CA-81534). Disclosures: No relevant conflicts of interest to declare.

Description: Apoptosis-based therapies have been used as a powerful weapon against hematologic malignancies (Reed JC et al, Blood 2005 15; 408-418). One strategy for attacking cancer is to overcome its resistance to apoptosis via specific targeting molecules. In this study, we are focusing on protein tyrosine kinase 7 (PTK7) as a new target for cancer therapy. PTK7, also known as CCK4, is a member of transmembrane tyrosine kinase family proteins that has been implicated in cell migration, polarity and apoptosis. PTK7 is overexpressed in acute T-cell lymphoblastic leukemia cells, and a recent report indicated that PTK7 expression was associated with poor prognosis. However, the mechanism by which PTK7 elicits its anti-apoptotic effects and directly promotes tumor proliferation has not yet been clarified. In addition, no data has been reported regarding the relationship between doxorubicin resistance and PTK7 expression. In this study, we investigated the role of PTK7 in chemotherapy resistance. Two different shRNA lentiviruses targeting PTK7 were stably infected in Jurkat cells (Jurkat tet-shPTK7#1 and #2), and gene knockdown assays against PTK7 were performed. In the presence of doxycycline, we observed a decrease in the viability of PTK7 knockdown cells following stimulation with αFAS-Ab (Jurkat tet-shPTK7#1: 28.6% ± 5.2%, #2: 8.7% ± 0.3%) compared to control cells (considered to be 100%). Similar results of decreased cell viability were also obtained with TRAIL stimulation (Jurkat tet-shPTK7#1: 22.5% ± 5.9%, #2: 14.7% ± 9.8%) compared to control cells (considered to be 100%). Furthermore, we observed a decrease in the viability of PTK7 knockdown cells following treatment with Doxorubicin compared to control cells treated with scramble siRNA. The reduction of cell viability in PTK7 knockdown cells could not be attributed to PTK7 knockdown itself since annexin-V and PI staining showed limited apoptotic changes in Jurkat tet-shPTK7 cells. These changes were also not the result of Fas receptor (CD95) and Trail receptor (DR4, DR5) expression up-regulation as flow cytometric analysis revealed no differences in the expression rates of Fas receptor and Trail receptor between scramble control cells and PTK7 knockdown cells. Very interestingly, PTK7 knockdown had no effect on viability when non-cancer cell lines were stimulated with Fas, TRAIL and Doxorubicin, suggesting that the synergistic effect of PTK7 knockdown and TNF family cytokine treatment is cancer specific. Next, to identify interaction partners of the PTK7 cytoplasmic domain (PTK7-cyto), we performed a yeast two-hybrid assay, and showed that FADD-DD was the only molecule found to bind to PTK7-cyto in both LEU2 reporter and LacZ reporter assays. This protein interaction was further verified by co-immunoprecipitation assays. Finally, to demonstrate the activation of Caspase-8 and PARP, which is induced by TNF-family death receptors, immunoblotting was performed. The shRNA-mediated silencing of PTK7 in Jurkat cells indeed increased Fas- and TRAIL-induced processing of caspase-8 and PARP compared to control RNA. These data suggest that PTK7 appears to be important for a proximal step in Fas and TRAIL signaling, which is consistent with the selective sensitization of tumor cells to the extrinsic apoptosis pathway. Together, our findings reveal an unexpected role for PTK7 in terms of death receptor-mediated apoptosis, suggesting that PTK7 could provide a new target for cancer therapy. Disclosures No relevant conflicts of interest to declare.

Description: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive hematological malignancy associated with the retrovirus human T-cell lymphotropic virus type I. Clinical outcomes of currently available chemotherapies are generally inferior with extremely poor prognosis. Previous studies have recently utilized next generation sequencing technology for the identification of mutated genes that may be pivotal in the pathogenesis of ATLL. However, the identification of indispensable genes for the proliferation and/or survival of ATLL cells remains a formidable challenge due to the complexity of genomic/epigenetic alterations in the ATLL genome. To investigate previously undescribed therapeutic targets in ATLL, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening to identify genetic vulnerabilities in ATLL cells. Three ATLL cell lines were transduced with lentiviral construct for Cas9 nuclease, followed by lentiviral delivery of the human CRISPR Brunello pooled library (Addgene 73178) of 76,456 single-guide RNAs targeting 19,144 protein-coding genes to cause DNA double-stranded cleavage by the Cas9 nuclease and loss-of-function of the respective genes. Compared with the control cell lines, 23 essential genes, including BATF3 (which we previously discovered by shRNA library screening; Nakagawa et al., Cancer Cell. 34:286-297. 2018) and novel genes (CDK6, JUNB, STAT3, and CCND2) were identified to be involved in ATLL cell proliferation and/or survival. Among these, CDK6 (cyclin-dependent kinase 6), a critical regulatory serine/threonine kinase that forms heterodimers with D-type cyclins, had the best score. The CDK6/D-type cyclin complex regulates E2F transcription factors through the phosphorylation of Rb (retinoblastoma protein), resulting in G1-S transition of the cell cycle. Utilizing publicly available microarray data from peripheral T-cell lymphoma patients, we demonstrated the higher expression of CDK6 in ATLL than that of the other subtypes of T-cell lymphomas, which prompted us to focus on CDK6 as a therapeutic molecular target in ATLL. In confirmatory experiments, two sgRNAs targeting the coding sequences of CDK6 exhibited strong toxicity in five ATLL cell lines in a temporal fashion, which was mediated by G1 cell arrest and partially through apoptosis. We confirmed on-target effect of the sgCDK6 by successfully rescuing cells from toxicity using retroviruses expressing sgRNA-resistant CDK6 cDNA in two ATLL cell lines. The knockout of CDK6 and decrease in the level of phosphorylated Rb were confirmed by immunoblot of sgCDK6-transduced ATLL cell lines. Collectively, the data showed an essential role for CDK6 in cellular proliferation and survival in ATLL. Of the 19,144 genes examined, CDK6 was considered the best vulnerable target for ATLL; therefore, we extended our analysis to evaluate the pharmacological inhibition of CDK6 in ATLL cells. Palbociclib, FDA-approved CDK4/6 inhibitor for breast cancer, was toxic in 11 ATLL cell lines and in four primary ATLL cells but the range of IC50 values were relatively broad (9-6500 nM) among ATLL lines. Because aproximately 20% of ATLL patients carry genetic alteration in a cell cycle/apoptosis regulator TP53 gene, we hypothesized that TP53 alteration may affect the sensitivity of ATLL cells to palbociclib. First, we assessed TP53 status of ATLL cell lines by Sanger sequencing and immunoblotting and showed that six TP53-altered ATLL cell lines exhibited significantly higher IC50 for palbociclib compared with five TP53-intact ATLL cell lines (p

Description: Abstract 521 DOCK180, an archetype CDM family protein and a guanine nucleoside exchange factor (GEF), plays a pivotal role in the regulation of cell motility and phagocytosis through the activation of Rac small GTPase. The expression of DOCK180 is ubiquitous except in hematopoietic cells, and a recent report indicated that DOCK180 was also found in dendritic cells (DCs) during their differentiation from CD14 positive monocytes (Fujimoto et al. ASH 2005). Although a knockout study of DOCK2, a hematopoietic cell-specific homolog of DOCK180, demonstrated the essential role of DOCK2 in murine lymphocyte motility and chemotaxis, no involvement of DOCK2 in DCs derived from monocytes has yet been observed. In addition, no data have so far been reported regarding the role of DOCK2 and DOCK180 in the motility of human DCs. In this study, we investigated the role of DOCK180, as well as DOCK2, in the chemotaxis of human DCs derived from monocytes. DCs were prepared by culturing the isolated monocytes from adult volunteers at the Hokkaido Blood Bank in the presence of GM-CSF and IL-4 for 7 days, and gene knock-down assays against DOCK180 or DOCK2 using siRNA were performed. Real time reverse transcriptase-polymerase chain reaction revealed the expression levels of DOCK180 and DOCK2 mRNA in DOCK180 knock-down and DOCK2 knock-down DCs to be almost half of those in DCs transfected with the control random oligonucleotide (control DCs). To determine the role of DOCK2 and DOCK180 in the migration evoked by CXCL12, transwell chemotaxis assays using these knock-down DCs were performed. The migration rates of DOCK180 knock-down DCs and DOCK2 knock-down DCs were significantly reduced to 46±15% and 47±18% compared to those of control DCs (considered to be 100%), respectively. We also found a reduced migration rate that was induced by another chemokine, CCL19, in both DOCK180 and DOCK2 knock-down DCs (34±5% and 47±12%, respectively). Next, we generated DOCK180/DOCK2 double knock-down DCs using the same siRNA technique and again performed the transwell migration assay. Interestingly, the double knock-down DCs showed additive reduction in their migration in response to CXCL12 and CCL19 compared to each single knock-down (19±2% and 15±6%, respectively). These reductions did not come from the down-regulation of chemokine receptors, since the flow cytometric analysis revealed no differences in the expression rates of CXCR4 and CCR7 in each type of DCs. Finally, to demonstrate the activation of Rac as a result of chemokine stimulation via DOCK2 and DOCK180, each of the knock-down DC lines were incubated with CXCL12, and a pull-down assay for Rac was performed. When control DCs were stimulated with CXCL12, the levels of the GTP-bound form of Rac were rapidly elevated within 5 seconds after the stimulation, and reached a peak after 15 seconds. This elevation of the GTP-bound form of Rac was significantly reduced in both DOCK180 or DOCK2 single knock-down DCs. Moreover, consistent with data from the chemotaxis study, very limited activation of Rac in the double knock down DCs was seen. These data demonstrated that DOCK180 and DOCK2 function as significant GEFs for Rac. Taken together, our data demonstrated that both DOCK180 and DOCK2 in human DCs derived from monocyte play a pivotal role in the chemotaxis in response to CXCL12 and CCL19, which do not correlate with the findings in murine DCs. Disclosures: No relevant conflicts of interest to declare.