ALBERT

Description: Translation of small interfering RNA (siRNA)–based approaches into practical therapeutics is limited because of lack of an effective and cell-specific delivery system. Herein, we present a new method of selectively delivering siRNA to dendritic cells (DCs) in vivo using CD40 siRNA-containing immunoliposomes (siILs) that were decorated with DC-specific DEC-205 mAb. Administration of CD40 siILs resulted in DC-specific cell targeting in vitro and in vivo. On treatment with CD40 siILs, the expression of CD40 in DCs, as well allostimulatory activity was inhibited. In vivo administration resulted in selective siRNA uptake into immune organs and functional immune modulation as assessed using a model antigen. In conclusion, this is the first demonstration of DC-specific siRNA delivery and gene silencing in vivo, which highlights the potential of DC-mediated immune modulation and the feasibility of siRNA-based clinical therapy.

Description: Abstract 1034 Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a therapeutically recalcitrant malignancy that accounts for approximately 15% of pediatric and 25% of adult ALL cases. In leukemia, cancer stem cells constitute a relatively rare population of tumor cells that play a key role in cancer propagation and, like adult stem cells, have enhanced self-renewal potential. A previous report showed that following in vitro culture, CD34+/CD4- and CD34+/CD7- subfractions of T-ALL marrow were enriched for leukemia stem cells (LSC) capable of engrafting leukemia in nonobese diabetic/severe combined immune deficient mouse (NOD/SCID). However, difficulties in maintaining primary cultures of leukemia cells hampered investigations into the biology of T-ALL underscoring the need for a direct transplantation model to characterize human LSC in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL. Experimental Procedures: Quantitative RT-PCR of NOTCH target gene expression and NOTCH mutation DNA sequencing analysis was performed on human CD34+ cells from T-ALL patient samples (n =12). To develop a humanized mouse model of T-ALL, CD34+ progenitors were lentivirally transduced with GFP-Luciferase Fusion protein (GLF) and transplanted intrahepatically into neonatal T, B, and NK cell deficient mice. In some experiments, FACS purified CD34+ subpopulations were transplanted at limiting dilution, including CD34+CD38+CD2+Lin- cells. Leukemic engraftment was monitored by in vivo bioluminescence imaging and analyzed by FACS detection of human CD34+ cells in liver, bone marrow, spleen and thymus when mice were sacrificed at 8–10 weeks post-transplant. NOTCH1 target gene expression was analyzed by q-RT-PCR in human CD34+ cells derived from engrafted tissues and NOTCH mutation analysis was performed by DNA sequencing on the same population. To assay LSC self-renewal, engrafted human CD34+ cells from bone marrow were transplanted into secondary and tertiary recipients. In serially transplanted mice, NOTCH1 target gene expression, NOTCH1 receptor expression was analyzed by FACS and NICD expression was assessed in the bone marrow by immunohistochemistry. Results: Q-RT-PCR data showed that NOTCH1, HES1 and c-MYC expression correlated with NOTCH 1 mutation status as well as the emergence of a CD34+CD2+Lin- population not evident in normal cord blood. We transplanted 12 T-ALL patient samples with detectable Notch1 expression and 100% of samples engrafted RAG 2-/- gamma c-/- mice. Transplanted LSC could be tracked for 10 weeks after transplant by in vivo bioluminescent imaging while Lin+ engraftment declined. Human CD34+/CD45+ cells, CD45+/CD34+/CD38+/Lin−/CD2+ cells were found in the bone marrow, thymus, spleen of the engrafted mice at 9–10 weeks post transplant or the end of dosing. Finally, human CD34+ cells engrafted secondary and tertiary recipients with T-ALL demonstrating their propensity for self-renewal and differentiation. Notch1 target gene and Hes1 expression was higher in patients with Notch1 mutation identified by sequencing. Conclusion: Serially transplantable candidate LSC retain high level NOTCH1 target gene expression and may be uniquely susceptible to targeted NOTCH1 receptor inhibition. Disclosures: Jamieson: Pfizer: Research Funding.

Description: Backgroud: Cardiac toxicity is a life-threatening complication in elderly patients with lymphoma, which lead to a delay or premature termination of chemotherapy. Methods: A total of 462 consecutive patients with diffuse large B-cell lymphoma over 60 years old between 2007 and 2017 were reviewed. Of these, 87 patients were excluded from the study. Finally, 375 lymphoma patients were included. Data about general information, clinical feature, laboratory examination, pathological results, therapeutic methods and cardiac toxicity were collected by case retrieval system. Cardiac toxicity was graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE). Results: The incidence of cardiac disorders was 5.3% (20/375). The median number of chemotherapy cycles before cardiac toxicity was 1 (range, 1-4). Ventricular arrhythmia was the most frequent cardiac disorder (n=6), followed by palpitations (n=4), left ventricular systolic dysfunction (n=3), heart failure (n=3), atrial fibrillation (n=2), myocardial infarction (n=1) and paroxysmal atrial tachycardia (n=1). At the end of treatment, grades 3 to 5 cardiac events were observed in 8 patients. In a multivariate Cox regression analysis, ECOG performance status ≥2 and history of cardiovascular disease were identified as risk factors for IP. The cumulative incidence of cardiac disorders were 2.3% (6/266) for patients without risk factors, 11.6% (11/95) for patients with 1 risk factors, and 21.4% (3/14), respectively. Conclusion: Cardiac toxicity is not rare in elderly patients with lymphoma, and a comprehensive management strategy is needed. Disclosures Song: Peking University Cancer Hospital (Beijing Cancer Hospital): Employment. Zhu:Beijing Cancer Hospital: Employment.

Description: Introduction: Extranodal natural killer/T-cell lymphoma (ENKTL), which is a very aggressive disease with poor prognosis, is prevalent in East Asian populations and accounts for 5-10% of all lymphomas in the Chinese population. The pathogenesis and molecular characteristics of different subgroups of ENKTL have largely remained elusive. Methods: Samples from 12 patients, aged 13 to 79, who were diagnosed with ENKTL in southwest China and received chemotherapy as first-line therapy, were collected for whole-exome sequencing (WES). Samples included 5 relapsed/refractory (rel/ref) patients and 7 treatment effective patients. The inclusion criteria for rel/ref group were that the patient progressed after 2 cycles of standard or conventional first-line treatment, or failed to achieve complete remission after 4 cycles (complete response (CR), or CR after treatment, but relapsed within 1 year after treatment). The inclusion criteria for the treatment effective group were that patient achieved CR after treatment, confirmed by long-term survival and no recurrence found after follow-up. In the rel/ref group, 3 cases occurred in the nasal cavity and 2 cases occurred outside the nasal cavity. In the treatment effective group, 3 cases occurred in the nasal cavity and 4 cases occurred outside the nasal cavity. The median overall survival (OS) in the rel/ref group and treatment effective group was 36 weeks and 80 weeks, respectively. Genomic DNA of tumors was extracted from the formalin-fixed, paraffin-embedded (FFPE) samples by using QIAamp DNA FFPE Tissue Kit. Along with virus infection status, somatic genomic alterations, including single nucleotide variations (SNV), short and long insertions and deletions (Indel), copy number variations (CNV), and gene rearrangements, were analyzed. Results: Common gene mutations (frequencies ≥25%) that occurred in both the rel/ref and treatment effective group included TP53, CD274, CSMD2, CUL9, EPPK1, PCDHA6, PCSK5, PKHD1, SDK1, STAT3 and TNXB. Relatively specific genes to the rel/ref group were ABCA13, COL22A1, NOTCH1, NDN, OBSL1, PPFIA2, SDK2, ZFP36L2, and ZNF860. In addition, we found that the most frequently mutated genes were different in patients with ENKTL occurring in the nasal cavity compared to those outside the nasal cavity. The high frequency mutated genes relatively specific to ENKTL in the nasal cavity were TP53 (50% vs. 17%), PKHD1 (50% vs. 0%), SDK1 (50% vs. 0%) and TNXB (50% vs. 0%), while in ENKTL outside the nasal cavity, EPPK1 (0% vs. 50%) was the most mutated gene. Meanwhile, we detected the wildly concerned biomarker CD274 (33% vs. 17%) and CHD8 (33% vs. 0%) in ENKTL occurring in the nasal cavity, and STAT3 (17% vs. 33%) in ENKTL outside the nasal cavity. Furthermore, we revealed that gene rearrangements were more common in the treatment effective group than in the rel/ref group (43% vs. 20%, respectively). A CD274 rearrangement and two TARP rearrangement were detected in 3/7 treatment effective patients, however, in the rel/ref ENKTL group, only a CD274 rearrangement was found. In this cohort, one ENKTL patient (1/12) exhibited a high tumor mutational burden (TMB) (15.4 Muts/Mb). Conclusion: Our findings revealed the molecular characteristics of different ENKTL subgroups, especially in the rel/ref subgroup, which might provide useful information for targeted/immunotherapy of ENKTL patients. Disclosures No relevant conflicts of interest to declare.

Description: Bone homeostasis is regulated by a delicate balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclastogenesis is controlled by the ratio of receptor activator of NF-κB ligand (RANKL) relative to its decoy receptor, osteoprotegerin (OPG). The source of OPG has historically been attributed to osteoblasts (OBs). While activated lymphocytes play established roles in pathological bone destruction, no role for lymphocytes in basal bone homeostasis in vivo has been described. Using immunomagnetic isolation of bone marrow (BM) B cells and B-cell precursor populations and quantitation of their OPG production by enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase–polymerase chain reaction (RT-PCR), cells of the B lineage were found to be responsible for 64% of total BM OPG production, with 45% derived from mature B cells. Consistently B-cell knockout (KO) mice were found to be osteoporotic and deficient in BM OPG, phenomena rescued by B-cell reconstitution. Furthermore, T cells, through CD40 ligand (CD40L) to CD40 costimulation, promote OPG production by B cells in vivo. Consequently, T-cell–deficient nude mice, CD40 KO mice, and CD40L KO mice display osteoporosis and diminished BM OPG production. Our data suggest that lymphocytes are essential stabilizers of basal bone turnover and critical regulators of peak bone mass in vivo.

Description: The low-density lipoprotein (LDL) receptor–related protein (LRP) has a well-established role in the hepatic removal of atherogenic apolipoprotein E (APOE)–rich remnant lipoproteins from plasma. In addition, LRP recognizes multiple distinct pro- and antiatherogenic ligands in vitro. Here, we investigated the role of hepatic LRP in atherogenesis independent of its role in removal of APOE-rich remnant lipoproteins. Mice that allow inducible inactivation of hepatic LRP were combined with LDL receptor and APOE double-deficient mice (MX1Cre+LRPflox/floxLDLR–/–APOE–/–). On an LDLR–/–APOE–/– background, hepatic LRP deficiency resulted in decreased plasma cholesterol and triglycerides (cholesterol: 17.1 ± 5.2 vs 23.4 ± 6.3 mM, P = .025; triglycerides: 1.1 ± 0.5 vs 2.2 ± 0.8 mM, P = .002, for MX1Cre+LRPflox/flox-LDLR–/–APOE–/– and control LRPflox/flox-LDLR–/–APOE–/– mice, respectively). Lower plasma cholesterol in MX1Cre+LRPflox/flox-LDLR–/–APOE–/– mice coincided with increased plasma lipoprotein lipase (71.2 ± 7.5 vs 19.1 ± 2.4 ng/ml, P = .002), coagulation factor VIII (4.4 ± 1.1 vs 1.9 ± 0.5 U/mL, P = .001), von Willebrand factor (2.8 ± 0.6 vs 1.4 ± 0.3 U/mL, P = .001), and tissue-type plasminogen activator (1.7 ± 0.7 vs 0.9 ± 0.5 ng/ml, P = .008) compared with controls. Strikingly, MX1Cre+LRPflox/floxLDLR–/–APOE–/– mice showed a 2-fold higher atherosclerotic lesion area compared with controls (408.5 ± 115.1 vs 219.1 ± 86.0 103μm2, P = .003). Our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its proatherogenic ligands.

Description: Human immunodeficiency virus type 1 (HIV-1) envelope protein gp41 mediates viral fusion with human host cells. The peptide segment T20/DP178, located in the C-terminus of the ectodomain of gp41, interacts with the N-terminal leucine zipper-like domain on gp41 to establish the fusogenic conformation of the virus. Synthetic T20/DP178 peptide is highly efficacious in inhibiting HIV-1 infection in vitro by disrupting the transformation of fusogenic status of viral gp41; thus, it has been proposed for clinical trial. We report that synthetic T20/DP178 is a chemoattractant and activator of human peripheral blood phagocytes but not of T lymphocytes. We further demonstrate that T20/DP178 specifically activates a seven-transmembrane, G-protein–coupled phagocyte receptor for N-formylated chemotactic peptides, formyl peptide receptor (FPR). Moreover, synthetic T20/DP178 analogs lacking N-terminal amino acids acted as FPR antagonists. Our results suggest that gp41 peptides regulate phagocyte function via FPR and identify a novel mechanism by which HIV-1 may modulate innate immunity.

Description: Abstract 1378 Tumorigenesis is a multi-step process and involves the silencing of tumor suppressor genes (TSGs) by genetic and/or epigenetic mechanisms. Aberrant hypermethylation of gene promoters is a major epigenetic mechanism associated with TSG silencing in cancer. To identify putative TSGs that might be epigenetically silenced in extranodal NK/T-cell lymphoma, nasal type (ENKL), a genome-wide screening was performed in a commonly deleted region 6q22.33-q23.2. PTPRK (protein tyrosine phosphatase, receptor type, kappa) was identified as the only gene out of 77 genes mapped to the 6q22.33-q23.2 region that was upregulated in four of five ENKL cell lines after treatment with the demethylation agent 5-aza-2' deoxycytidine (5-aza-dC). Further analysis by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) confirmed that the CpG island surrounding the transcriptional start site of PTPRK was methylated in ENKL cell lines not expressing PTPRK. Similarly, a significant correlation between methylation of the PTPRK promoter and downregulation of PTPRK mRNA and protein expression (p=0.019 and p=0.048, respectively) was detected in 39 primary ENKLs. PTPRK gene allelic loss was detected in 80% of cell lines and 47% of primary ENKLs. Functional analyses by in vitro assays showed that the re-expression of PTPRK by retroviral transduction in the PTPRK non-expressing NKYS cell line suppressed the size and number of colonies formed, led to a remarkable increase in the apoptotic cell population and cell cycle arrest at G0/G1 phase. Moreover, PTPRK re-expression substantially reduced the migration and invasion of NKYS cells. Conversely, the inhibitory effect of PTPRK was significantly decreased by partial shRNA knockdown of PTPRK expression in the PTPRK-expressing SNK-6 cell line. The re-expression of PTPRK in another PTPRK non-expressing YT cell line suppressed tumor growth and metastasis in a nude mouse xenograft model. Consistent with these in vitro and in vivo findings, clinicopathological correlation analysis showed that PTPRK silencing was detected mostly in the ENKL patients with advanced and metastatic disease. Examination of the PTPRK protein sequence revealed that the cytoplasmic domain possesses a consensus STAT3 binding domain (YXXQ). Re-expression of PTPRK in NKYS cells resulted in a significant decrease in the level of phospho-STAT3 (Tyr705), whereas PTPRK knockdown in SNK-6 cells resulted in an increased level of phospho-STAT3 (Tyr705). These data suggested that PTPRK dephosphorylates and regulates the oncoprotein STAT3. Overall, this study shows that PTPRK is a putative TSG in the 6q22.33-q23.2 region that is frequently deleted and epigenetically silenced in ENKL, and the loss of PTPRK expression promotes tumor growth via the aberrant constitutive activation of STAT3 in ENKL. Specific therapies aimed at targeting STAT3 warrants further exploration. Disclosures: No relevant conflicts of interest to declare.

Description: Human immunodeficiency virus type 1 (HIV-1) envelope protein gp41 mediates viral fusion with human host cells. The peptide segment T20/DP178, located in the C-terminus of the ectodomain of gp41, interacts with the N-terminal leucine zipper-like domain on gp41 to establish the fusogenic conformation of the virus. Synthetic T20/DP178 peptide is highly efficacious in inhibiting HIV-1 infection in vitro by disrupting the transformation of fusogenic status of viral gp41; thus, it has been proposed for clinical trial. We report that synthetic T20/DP178 is a chemoattractant and activator of human peripheral blood phagocytes but not of T lymphocytes. We further demonstrate that T20/DP178 specifically activates a seven-transmembrane, G-protein–coupled phagocyte receptor for N-formylated chemotactic peptides, formyl peptide receptor (FPR). Moreover, synthetic T20/DP178 analogs lacking N-terminal amino acids acted as FPR antagonists. Our results suggest that gp41 peptides regulate phagocyte function via FPR and identify a novel mechanism by which HIV-1 may modulate innate immunity.

Description: Key Points PTPRK binds to STAT3 and directly dephosphorylates phospho-STAT3 at Tyr705. Loss of PTPRK, located in the deleted 6q region, leads to STAT3 activation and contributes to nasal-type NK/T-cell lymphoma pathogenesis.