Monica Puppi

Molecular Sites of Regulation of Expression of the Rat Cationic Amino Acid Transporter Gene

Cat-1 is a protein with a dual function, a high affinity, low capacity cationic amino acid transporter of the y+ system and the receptor for the ecotropic retrovirus. We have suggested that Cat-1 is required in the regenerating liver for the transport of cationic amino acids and polyamines in the late G1 phase, a process that is essential for liver cells to enter mitosis. In our earlier studies we had shown that the cat-1 gene is silent in the quiescent liver but is induced in response to hormones, insulin, and glucocorticoids, and partial hepatectomy. Here we demonstrate that cat-1 is a classic delayed early growth response gene in the regenerating liver, since induction of its expression is sensitive to cycloheximide, indicating that protein synthesis is required. The peak of accumulation of the cat-1 mRNA (9-fold) by 3 h was not associated with increased transcriptional activity of the cat-1 gene in the regenerating liver, indicating post-transcriptional regulation of expression of this gene. Induction of the cat-1 gene results in the accumulation of two mRNA species (7.9 and 3.4 kilobase pairs (kb)). Both mRNAs hybridize with the previously described rat cat-1/2.9-kb cDNA clone. However, the 3′ end of a longer rat cat-1 cDNA (rat cat-1/6.5-kb) hybridizes only to the 7.9-kb mRNA transcript. Sequence analysis of this clone indicated that the two mRNA species result from the use of alternative polyadenylation signals. The 6.5-kb clone contains a number of AT-rich mRNA destabilizing sequences which is reflected in the half-life of the cat-1 mRNAs (90 min for 7.9-kb mRNA and 250 min for 3.4-kb mRNA). Treatment of rats with cycloheximide superinduces the level of the 7.9-kb cat-1 mRNA in the kidney, spleen, and brain, but not in the liver, suggesting that cell type-specific labile factors are involved in its regulation. We conclude that the need for protein synthesis for induction of the cat-1 mRNA, the short lived nature of the mRNAs, and the multiple sites for regulation of gene expression indicate a tight control of expression of the cat-1 gene within the regenerating liver and suggest that y+ cationic amino acid transport in liver cells is regulated at the molecular level.

Krüppel-like factor 4 (KLF4) promotes the survival of natural killer cells and maintains the number of conventional dendritic cells in the spleen

The development and survival of NK cells rely on a complex, spatiotemporal gene expression pattern regulated by specific transcription factors in NK cells and tissue‐specific microenvironments supported by hematopoietic cells. Here, we show that somatic deletion of the KLF4 gene, using inducible and lineage‐specific cre‐transgenic mice, leads to a significant reduction of NK cells (NK1.1+ TCR‐β−) in the blood and spleen but not in the BM, liver, or LNs. Functional and immunophenotypic analyses revealed increased apoptosis of CD27+/− CD11b+ NK cells in the spleen of KLF4‐deficient mice, although remaining NK cells were able to lyse tumor target cells and produce IFN‐γ. A normal recovery of adoptively transferred KLF4‐deficient NK cells in WT hosts suggested that the survival defect was not intrinsic of NK cells. However, BM chimeras using KLF4‐deficient mice as donors indicated that reduced survival of NK cells depended on BM‐derived hematopoietic cells in the spleen. The number of CD11chi DCs, which are known to support NK cell survival, was reduced significantly in the spleen of KLF4‐deficient mice, likely a result of a lower number of precDC progenitor cells in this tissue. Taken together, our data suggest that the pluripotency‐associated gene KLF4 is required for the maintenance of DCs in the spleen and consequently, survival of differentiated NK cells in this tissue.

Differential roles of KLF4 in the development and differentiation of CD8+ T cells

The transcription factor Krüppel-like factor 4 (KLF4) can activate or repress gene expression in a cell-context dependent manner. We have previously shown that KLF4 inhibits the proliferation of naïve CD8(+) T cells in vitro downstream of the transcription factor ELF4. In this work, we describe a novel role of KLF4 in the differentiation of CD8(+) T cells upon infection. Loss of KLF4 had minimal effect on thymic T cell development and distribution of mature T cells in the spleen, blood, and lymph nodes. KLF4-deficient naïve CD8(+) T cells also displayed normal homeostatic proliferation upon adoptive transfer into lymphopenic hosts. However, activation of KLF4-deficient naïve CD8(+) T cells by in vitro TCR crosslink and co-stimulation resulted in increased proliferation. Furthermore, naïve KLF4-deficient OT-I CD8(+) T cells generated increased numbers of functional memory CD8(+) T cells compared to wild type OT-I CD8(+) T cells co-injected in the same recipient in both primary and recall responses to Listeria monocytogenes-OVA. Collectively, our data demonstrate that KLF4 regulates differentiation of functional memory CD8(+) T cells while sparing development and homeostasis of naïve CD8(+) T cells.

The Transcription Factor E74-Like Factor 4 Suppresses Differentiation of Proliferating CD4+ T Cells to the Th17 Lineage

The differentiation of CD4+ T cells into different Th lineages is driven by cytokine milieu in the priming site and the underlying transcriptional circuitry. Even though many positive regulators have been identified, it is not clear how this process is inhibited at transcriptional level. In this study, we report that the E-twenty six (ETS) transcription factor E74-like factor 4 (ELF4) suppresses the differentiation of Th17 cells both in vitro and in vivo. Culture of naive Elf4−/− CD4+ T cells in the presence of IL-6 and TGF-β (or IL-6, IL-23, and IL-1β) resulted in increased numbers of IL-17A–positive cells compared with wild-type controls. In contrast, the differentiation to Th1, Th2, or regulatory T cells was largely unaffected by loss of ELF4. The increased expression of genes involved in Th17 differentiation observed in Elf4−/− CD4+ T cells suggested that ELF4 controls their programming into the Th17 lineage rather than only IL-17A gene expression. Despite normal proliferation of naive CD4+ T cells, loss of ELF4 lowered the requirement of IL-6 and TGF-β signaling for IL-17A induction in each cell division. ELF4 did not inhibit Th17 differentiation by promoting IL-2 production as proposed for another ETS transcription factor, ETS1. Elf4−/− mice showed increased numbers of Th17 cells in the lamina propria at steady state, in lymph nodes after immunization, and, most importantly, in the CNS following experimental autoimmune encephalomyelitis induction, contributing to the increased disease severity. Collectively, our findings suggest that ELF4 restrains Th17 differentiation in dividing CD4+ T cells by regulating commitment to the Th17 differentiation program.

Transcription factor ELF4 promotes development and function of memory CD8+ T cells in Listeria monocytogenes infection

Most differentiated CD8+ T cells die off at the end of an infection, revealing two main subsets of memory T cells — central and effector memory — which can be found in lymphoid tissues or circulating through nonlymphoid organs, respectively. The cell intrinsic regulation of the differentiation of CD8+ T cells to effector and central memory remains poorly studied. Herein, we describe a novel role of the ETS transcription factor ELF4 in the development and function of memory CD8+ T cells following infection with Listeria monocytogenes. Adoptively transferred Elf4−/− naïve CD8+ T cells produced lower numbers of effector memory CD8+ T cells despite a normal pool of central memory. This was caused by suboptimal priming and decreased survival of CD8+ T cells at the peak of response while enhanced Notch1 signaling and upregulation of eomesodermin correlated with “normal” development of Elf4−/− central memory. Finally, loss of ELF4 impaired the expansion of both central and effector memory CD8+ T cells in a recall response by also activating Notch1 signaling. Altogether, ELF4 emerges as a novel transcriptional regulator of CD8+ T‐cell differentiation in response to infection.

Inactivation of KLF4 promotes T-cell acute lymphoblastic leukemia and activates the MAP2K7 pathway.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a high incidence of relapse in pediatric ALL. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate the onset of leukemia and worsen disease progression are largely unknown. Here, we show that the gene encoding the transcription factor KLF4 is inactivated by DNA methylation in children with T-ALL. In mice, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL by enhancing the G1-to-S transition in leukemic cells and promoting the expansion of leukemia-initiating cells. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7. Our results showed that in murine and pediatric T-ALL, loss of KLF4 leads to aberrant activation of MAP2K7 and of the downstream effectors JNK and ATF2. As a proof-of-concept for the development of a targeted therapy, administration of JNK inhibitors reduced the expansion of leukemia cells in cell-based and patient-derived xenograft models. Collectively, these data uncover a novel function for KLF4 in regulating the MAP2K7 pathway in T-ALL cells, which can be targeted to eradicate leukemia-initiating cells in T-ALL patients.

G0S2 modulates homeostatic proliferation of naïve CD8 + T cells and inhibits oxidative phosphorylation in mitochondria

Since its discovery, diverse functions have been attributed to the G0/G1 switch gene 2 (G0S2), from lipid metabolism to control of cell proliferation. Our group showed for the first time that G0S2 promotes quiescence in hematopoietic stem cells by interacting with and retaining nucleolin around the nucleus. Herein, we report the role of G0S2 in the differentiation and function of CD8+ T cells examined in mice with an embryonic deletion of the G0s2 gene. G0S2 expression in naïve CD8+ T cells decreased immediately after T‐cell receptor activation downstream of the mitogen‐activated protein kinase, calcium/calmodulin, phosphatidylinositol 3′‐kinase and mammalian target of rapamycin pathways. Surprisingly, G0S2‐null naïve CD8+ T cells displayed increased basal and spare respiratory capacity that was not associated with increased mitochondrial biogenesis but with increased phosphorylation of AMP‐activated protein kinase α. Naïve CD8+ T cells showed increased proliferation in response to in vitro activation and in vivo lymphopenia; however, naïve CD8+ T cells expressing the OT‐1 transgene exhibited normal differentiation of naïve cells to effector and memory CD8+ T cells upon infection with Listeria monocytogenes in a wild‐type or a G0s2‐null environment, with increased circulating levels of free fatty acids. Collectively, our results suggest that G0S2 inhibits energy production by oxidative phosphorylation to fine‐tune proliferation in homeostatic conditions.

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.