Daniel Lacorazza

Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication

Zika virus (ZIKV) is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Although ZIKV has been predominately associated with a mild or asymptomatic dengue-like disease, its appearance in the Americas has been accompanied by a multi-fold increase in reported incidence of fetal microcephaly and brain malformations. The source and mode of vertical transmission from mother to fetus is presumptively transplacental, although a causal link explaining the interval delay between maternal symptoms and observed fetal malformations following infection has been missing. In this study, we show that primary human placental trophoblasts from non-exposed donors (n = 20) can be infected by primary passage ZIKV-FLR isolate, and uniquely allowed for ZIKV viral RNA replication when compared to dengue virus (DENV). Consistent with their being permissive for ZIKV infection, primary trophoblasts expressed multiple putative ZIKV cell entry receptors, and cellular function and differentiation were preserved. These findings suggest that ZIKV-FLR strain can replicate in human placental trophoblasts without host cell destruction, thereby serving as a likely permissive reservoir and portal of fetal transmission with risk of latent microcephaly and malformations.

Abstract LB-181: KLF4 suppresses T-cell acute lymphoblastic leukemia by inhibiting the stress kinase MAP2K7 pathway

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with the highest incidence of relapse of any pediatric ALL. A minimal two-hit model of leukemogenesis suggests that an initial genetic driver transforms hematopoietic progenitor cells into LICs, whereas a secondary genetic alteration would endow LICs with proliferative and survival advantages. Although most T-ALL patients exhibit activating mutations in NOTCH1 , the cooperating genetic events required to accelerate onset of leukemia and worsen disease progression are largely unknown. Here, we show that low levels of the transcription factor KLF4 in children with T-ALL were associated with methylation of its promoter. Consistent with a tumor suppressor function, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL in mice by enhancing the G1-to-S transition and promoting the expansion of leukemia-initiating cells that are responsible for chemoresistance and relapses. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7, and thus loss of KLF4 activates MAP2K7 and downstream effector JNK both in murine model of T-ALL and lymphoblasts from pediatric patients with T-ALL. Furthermore, pharmacological inhibition of JNK reduced leukemia burden in a xenograft model of human T-ALL and small molecule inhibitors exhibited anti-leukemic properties in patient-derived xenograft cells. In summary, our findings demonstrate a novel tumor suppressor function of KLF4 in a T-ALL mouse model and in pediatric T-ALL and support a model of leukemia inhibition by repression of the stress kinase MAP2K7 and its downstream targets JNK, c-JUN, and ATF2. In addition, our study provides proof-of-principle pre-clinical data supporting JNK inhibition as a potential targeted therapy for T-ALL and prompts future studies in high-risk T-ALL patients with refractory and relapsed disease. Citation Format: Ye Shen, Chun Shik Park, Koramit Suppipat, Toni-Ann Mistretta, Monica Puppi, Terzah Horton, Karen Rabin, Daniel Lacorazza. KLF4 suppresses T-cell acute lymphoblastic leukemia by inhibiting the stress kinase MAP2K7 pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-181.

Abstract B19: Inactivation of KLF4 in T cell acute lymphoblastic leukemia promotes the expansion of leukemia cells by activating the Map2k7/Jnk pathway.

Acute lymphoblastic leukemia is the most common hematological malignancy in pediatric patients, and disease relapse is the leading cause of cancer-associated death in children. Despite steadily improved outcomes in patients with newly diagnosed disease, little progress has been made to treat relapse leukemia and to reduce the incidence of relapse by increasing the cure rates of frontline therapy. Targeted therapy is currently not available for T-ALL and the development of novel agents requires a better understanding of how leukemia-initiating cells (LIC) are maintained. In contrast to normal blood cells, we found that the transcription factor KLF4 is significantly downregulated in pediatric T-ALL. Next-generation bisulfite sequencing revealed that the KLF4 promoter is hypermethylated in lymphoblasts from T-ALL patients but not in normal bone marrow cells, T cells, and pediatric B-ALL. Deletion of the Klf4-floxed gene with Vav-iCre transgene and transplantation of Klf4 Δ / Δ bone marrow cells transduced with the Notch1-L1601P-ΔP mutant resulted in a significant acceleration of T-ALL due to a 9-fold expansion of LIC and increased G1-S transition in leukemic T cells. A combined analysis of global gene expression and genome-wide binding revealed that KLF4 directly represses the dual specificity mitogen-activated protein kinase 7 (Map2k7) and thus Klf4 Δ / Δ T-ALL mice exhibit increased Map2k7 expression. Most remarkably, T-ALL cells from both mouse model and pediatric patients showed elevated levels of total and phosphorylated Map2k7 and subsequent activation of downstream targets JNK, c-Jun, and ATF2. Finally, we tested several JNK inhibitors in T-ALL cell lines, primary patient samples, and pre-clinical xenograft models of T-ALL and confirmed that JNK inhibition blocks ATF2 activation and controls expansion of leukemia cells, validating the Map2k7/Jnk pathway as a selective target for T-ALL therapy. Thus, KLF4 emerges as a novel tumor suppressor in T-ALL and Jnk inhibition as a novel therapeutic approach for adjunctive therapy in T-ALL patients with refractory or relapse disease. Citation Format: Ye Shen, Koramit Suppipat, Chun Shik Park, Toni-Ann Mistretta, Terzah Horton, Karen Rabin, Daniel Lacorazza. Inactivation of KLF4 in T cell acute lymphoblastic leukemia promotes the expansion of leukemia cells by activating the Map2k7/Jnk pathway.. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B19.

Sa1742 Microbiome and Host Factors Communicate Protection Against Acute Murine Colitis Following Omega-6 Fatty Acid Induced Transient Pediatric Obesity

Background: Dietary influences may affect microbiota composition and host immune responses at the intestinal surface. These nutritionally induced biologic component changes may modulate propensity towards inflammatory bowel diseases (IBD), including Crohn disease (CD) and ulcerative colitis (UC). IBD present most commonly in young adults, indicating childhood as a potentially vulnerable developmental period for disease pathogenesis. Dietary omega-6 fatty acids (ω-6) have been associated with UC in prospective human epidemiologic studies. However, the critical developmental period, when ω-6 consumption may induce UC is not known. We examined the prolonged effects of transient high ω-6 diet during pediatric development in a murine colitis model. Methods: C57Bl/6J mice received high (40% caloric content [cc]), or control (12% cc) ω-6 diet from postnatal day 30 (P30) to P80, then reversed to control diet for 40 days (P120). Body composition was examined by qMRI. Colitis was induced by dextran sulfate sodium (DSS). The severity of colitis was assessed by weight loss and histological scoring. Selective microbiome effects on DSS colitis were studied following fecal transplantation into germ-free mice. The mucosal microbiome was interrogated by next-generation pyrosequencing of the bacterial 16S rRNA gene. Microarrays to study DNA methylation and gene expression from colonic mucosa were utilized. T cell populations in mesenteric lymph nodes (MLNs) and spleen were analyzed by flow cytometry. Plasma of 12 treatment Naive (TN) UC, 11 TN CD and 10 controls were used for quantification of circulating CXCL13 levels by ELISA. Results: Mice transiently became obese on high ω-6 diet. Surprisingly, those were protected against DSS colitis. Protection against colitis was fat type and dietary reversal dependent. Germ-free mice receiving cecal content from the transiently obese mice were protected against colitis. No significant colonic mucosal DNA methylation or gene expression changes were detected. In the meantime, the number of CD4+ cells was decreased in both the spleens and MLNs of the high ω-6 reversed mice. MLN Cxcr5+/CD4+ cells, specifically, were decreased following transient obesity. AntiCxcl13 (the ligand of Cxcr5) antibody treatment decreased DSS induced histological colitis severity. Elevated CXCL13 concentrations (CD: 1.8-fold, p=0.0077; UC: 1.9-fold, p=0.056) were found in the serum of human pediatric IBD patients. Summary: Loss of ω-6 diet induced pediatric obesity protected against acute colitis inmice. This phenotype was communicated by prolongedmicrobiome changes and associatedwith immune organ compositionmodification. The Cxcr5-Cxcl13 pathway was indicated to be an important host factor in modulating DSS colitis severity following the dietary reversal. Our human serologic observations supported the translational relevance of our findings.