Par-4 is a leucine zipper domain protein that induces apoptosis on its own in certain cancer cells and in Ras-transformed cells, but not in normal or immortalized cells. Par-4 induces apoptosis by activation of the Fas death receptor pathway and co-parallel inhibition of NF-B transcription activity. Cells that are resistant to apoptosis by Par-4 alone, however, are greatly sensitized by Par-4 to the action of other pro-apoptotic insults such as growth factor withdrawal, TNF, ionizing radiation, intracellular calcium elevation, or those involved in neuronal degeneration such as Alzheimer's, Parkinson's, Huntington's and Stroke. Previous studies have suggested that the apoptosis-sensitization potential of Par-4 is dependent upon inhibition of PKC or WT1 cell survival function by direct interaction between the leucine zipper domain at the carboxy-terminus of Par-4 and the zinc finger domains of PKC or WT1. In this study, I performed structure-function analysis using GFP-fusion proteins and deletion mutants to identify the functional localization and domains of Par-4 that are essential for apoptosis induction. My findings suggest that apoptosis by Par-4 is dependent on its translocation to the nucleus for induction of apoptosis. A bipartite nuclear localization signal sequence corresponding to amino acids 137-155 was necessary for nuclear translocation of Par-4. Importantly, the core residues 137-204 in the center part of Par-4 were necessary and sufficient to induce Fas pathway activation, inhibition of nuclear NF-B transcription activity and apoptosis. These findings imply that binding of Par-4 via its leucine zipper domain to other proteins is dispensable for apoptosis by Par-4.

Prostate Apoptosis Response-4 (Par-4) is a conserved and ubiquitous tumor-suppressor factor which can selectively induce apoptosis in tumor cells, while leaving normal cells unaffected. While Par-4 is well established as a tumor-suppressor, there have yet been no formal investigations as to whether it has a physiologic role in normal tissues. Early observations of Par-4 knockout mouse lines yielded that the adult mice displayed significant weight gain and fat accumulation compared to their wild-type counterparts while on a conventional chow diet. Interestingly, obese mouse and human subjects were found to exhibit reduced expression of Par-4 in adipose tissue as well as lower levels of secreted Par-4 in their plasma, compared to samples collected from lean human subjects. Subsequent in vitro experiments would show that loss of Par-4 has significant impact upon adipogenesis. Mechanistically, Par-4 loss during adipogenesis in cell culture correlated inversely with expression of the adipogenic transcription factor PPARγ. Subsequent experiments would demonstrate that Par-4 transcriptionally represses PPARγ at the promoter level. Thereby, we conclude that Par-4 regulates adipogenesis and lipid accumulation through transcriptional repression of the PPARγ promoter. This research utilizes novel models and may be used as the basis for Par-4-mediated therapies for obesity and metabolic disease.

There are four mammalian SSB proteins (SSB-1 to -4), and these are characterized by a C-terminal SOCS box and central SPRY domain. The C-terminal SOCS box was first observed in proteins that were found to act as Suppressors of Cytokine Signalling and function by virtue of their SH2 domain. Other families containing the SOCS box motif were defined by the domains N-terminal to this, such as the ASBs (Ankyrin repeats), WSBs (WD40 repeats) and of course the SSBs (SPRY domains). This thesis describes a very broad investigation of the SSBs, a protein family about which very little was known. To begin with, functional investigation into the evolution of this family and analysis of murine SSB expression patterns was performed. This highlighted that the family was highly conserved and had differential expression in the mouse, suggestive of important, unique functional roles for the individual family members. The majority of work in the thesis then proceeds in three directions; (i) analysis of the SSB proteins in vivo, with genetic deletion of SSB-2 in the mouse, (ii) biochemically, with analysis of SSB binding partners, and (iii) structurally, with functional analysis of the structure of SSB-2.

O câncer de mama é a neoplasia de maior ocorrência na população feminina. É uma doença hormônio-dependente com etiologia complexa e multifatorial. O câncer de mama invasivo é uma das principais causas da morbidade e mortalidade de mulheres no mundo. O desenvolvimento e a progressão de câncer de mama estão associados com acúmulo de várias alterações genéticas e epigenéticas, que resultam na expressão gênica diferencial entre células normais e tumorais. A hipóxia pode ser considerada uma das características principais de tumores sólidos e uma força motriz para a sobrevida das células tumorais e progressão maligna. O gene supressor de tumor PAWR (PKC apoptosis WT1 regulator; também denominado PAR-4, prostate apoptosis response-4) tem importante papel na apoptose tanto pela via intrínseca quanto pela via extrínseca e pode ser considerado um alvo para a terapia seletiva de células tumorais uma vez que a expressão de PAR-4 aumenta a sensibilidade da maioria das células de câncer a um segundo estímulo apoptótico. Alguns estudos, incluindo os de nosso grupo, têm mostrado o envolvimento de PAR-4 em câncer de mama e seu papel como fator prognóstico e preditivo de resposta a quimioterapia. No entanto, pouco é conhecido sobre o papel funcional deste gene ou os mecanismos envolvidos na regulação da expressão de PAR-4 na glândula mamária e no processo de tumorigênese da mama. No presente trabalho investigamos os efeitos da hipóxia na expressão de PAR-4 nas linhagens celulares MCF10A, MCF7 e MDA-MB-231. Para isto, as células foram incubadas por períodos de 30 minutos, 1, 2, 6 ou 24 horas em ambiente controlado com 5 % CO2, 95% N2 e 0.2% O2. A expressão de Par-4 foi avaliada quanto aos transcritos, por PCR em Tempo Real e quanto à expressão protéica, por western blot. Observamos que a baixa concentração de oxigênio diminui a expressão do mRNA de PAR-4, em tempos de 6 e 24 horas, nas linhagens MCF10A e MCF7 e no tempo de 24h nas células MDA-MB-231. Já quando os níveis protéicos de Par-4 foram analisados não foi observada diferença significativa nessas células após exposição à condição de hipóxia. A expressão do gene NDRG1, a qual se encontra aumentada em condições de hipóxia, foi utilizada como controle. Nossos dados sugerem que a expressão dos transcritos do gene supressor de tumor PAR-4 é inibida pelos mecanismos celulares de sobrevivência celular ativados durante a hipóxia / Breast cancer has the highest incidence rate of all malignant neoplasias affecting women. It is a hormone dependent disease with complex and multifactorial etiology. Invasive breast cancer is one of the principal causes of women morbidity and mortality in the world. Breast cancer development and progression are associated with accumulation of various genetic and epigenetic alterations, which result in differential gene expression among normal and tumor cells. Hypoxia could be considered one of the principal hallmarks of solid tumors and the driving force for the tumor cell survival and malignant progression. The tumor suppressor gene PAWR (PKC apoptosis WT1 regulator; also known as PAR-4, prostate apoptosis response-4) has an important role in apoptosis either by intrinsic or extrinsic pathways and could be considered as a target for selective therapy of tumor cells. Some studies, including of our group, have shown the PAR-4 involvement in breast cancer and its role as prognostic and predictive factor in response to chemotherapy. However, little is known about functional role of this gene or the mechanisms involved in PAR-4 expression regulation in mammary gland and in breast tumorigenesis process. In the present study we evaluated the hypoxia effects on PAR-4 expression in MCF10A, MCF7 and MDA-MB-231 cell lines. Therefore, the cells were incubated for time periods of 30 minutes, 1, 2, 6 or 24 hours under controlled environment with 5 % CO2, 95% N2 e 0.2% O2. PAR-4 expression was evaluated for transcripts, by Real Time PCR, and for protein expression, by western blot. We observed that low oxygen concentration decreases PAR-4 mRNA expression, in periods of 6 and 24 hours, in MCF10A and MCF7 cell lines and in the period of 24 hours in MDA-MB-231 cells. However, when the Par-4 protein levels were evaluated, no significant difference was observed in these cells after incubation under hypoxic conditions. NDRG1 gene expression, which is increased under hypoxia, was used as a control. Our data suggest that the expression of tumor suppressor gene PAR-4 transcripts is inhibited by cell survival mechanisms activated during hypoxia

O câncer de mama é o tumor mais incidente e a principal causa de mortalidade entre as mulheres no mundo. Não diferente de tantos outros tipos tumorais, o câncer de mama carrega em sua história uma etiologia complexa, heterogênea e multifatorial. O gene pró-apoptótico PAWR (PKC apoptosis WT1 regulator; também denominado como PAR-4, Prostate Apoptosis Response-4) é conhecido por induzir seletivamente apoptose em uma grande variedade de células de câncer. O papel de Par-4 como supressor tumoral vem sendo bem estabelecido nos últimos anos. Entretanto, pouco tem sido explorado sobre o papel e os mecanismos envolvendo a função supressora de Par-4 em câncer de mama. Em estudos prévios do nosso grupo, foi possível demonstrar que a expressão reduzida de Par-4 está associada a um pior prognóstico em câncer de mama e que esta proteína pode ter um papel importante na morfogênese da glândula mamária. Além disso, a investigação em células MCF-7 demostrou que a expressão de Par-4 aumenta a sensibilidade das células ao tratamento com docetaxel. Com objetivo de entender melhor o papel de Par-4 em células tumorais de mama, nós investigamos o efeito da supressão de Par-4 nas células MDA-MB-231 in vitro e in vivo. A redução da expressão foi alcançada a partir de transfecção das células com vetores plasmidiais contendo shRNAs específicos para o transcrito de Par-4 e subsequentemente foram realizados ensaios funcionais. A expressão reduzida de Par-4 foi capaz de aumentar a capacidade de formação de colônias das células MDA-MB-231 em cultura. Além disso, as células MDA-MB-231 transfectadas com shRNA-Par-4 tiveram uma redução significativa da morte celular (fase sub-G0\\G1 do ciclo celular), em particular da morte por apoptose (Anexina-FITC/PI), após o tratamento com diferentes quimioterápicos. Em nosso estudo, as células MDA-MB-231-Controle tratadas com docetaxel apresentaram aumento nos níveis de Par-4 secretado, o que não foi observado nas células MDA-MB-231-shPar-4. Finalmente, nossos resultados in vivo sugerem que a expressão diminuída de Par-4 pode modular o crescimento tumoral em camundongos Balb/c NUDE. Em conjunto, nossos dados colaboram com o papel supressor de Par-4 já descrito na literatura e confirmam sua ação supressora em diferentes linhagens de câncer de mama / Breast cancer is the most incident tumor and the leading cause of mortality among women worldwide. Like other forms of cancer, breast cancer has a complex, heterogeneous and multifactorial etiology. The pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also known as PAR-4, Prostate Apoptosis Response-4) is known to selectively induce apoptosis in a wide variety of cancer cells. The role of Par- 4 as a tumor suppressor has been well established in recent years. However, little has been explored about the role and mechanisms of Par-4 suppressor function in breast cancer. Previous work from our research group demonstrated that the reduced expression of Par-4 is associated with a worse prognosis in breast cancer. Also an important role of Par-4 in the morphogenesis of the mammary gland was suggested. In addition, Par-4 overexpression in MCF-7 cells increased the sensitivity to docetaxel treatment. In order to better understand the role of Par-4 in breast tumor cells, we investigated the effect of Par-4 knock-down on MDA-MB-231 cells in vitro and in vivo. We performed shRNA-mediated Par-4 knockdown, and then carried out functional assays. The reduced expression of Par-4 was able to increase the colony formation capacity of MDA-MB-231 cells in culture. In addition, shRNA-Par-4 transfection in MDA-MB-231 cells led to a significant reduction of cell death (sub-G0/G1 cell cycle), particularly by apoptosis (Annexin-FITC/PI), after treatment with different chemotherapeutic agents. In our study, docetaxel-treated MDA-MB-231-Control cells showed increased levels of secreted Par-4, which was not observed in MDA-MB-231- shPar-4 cells. Finally, our in vivo results suggest that diminished Par-4 expression may modulate tumor growth in Balb/c NUDE mice. Taken together, our data support the suppressor role of Par-4 already described in the literature and confirm its suppressive action in different breast cancer cell lines

O câncer de mama é o tumor mais incidente e a principal causa de mortalidade entre as mulheres no mundo. Não diferente de tantos outros tipos tumorais, o câncer de mama carrega em sua história uma etiologia complexa, heterogênea e multifatorial. O gene pró-apoptótico PAWR (PKC apoptosis WT1 regulator; também denominado como PAR-4, Prostate Apoptosis Response-4) é conhecido por induzir seletivamente apoptose em uma grande variedade de células de câncer. O papel de Par-4 como supressor tumoral vem sendo bem estabelecido nos últimos anos. Entretanto, pouco tem sido explorado sobre o papel e os mecanismos envolvendo a função supressora de Par-4 em câncer de mama. Em estudos prévios do nosso grupo, foi possível demonstrar que a expressão reduzida de Par-4 está associada a um pior prognóstico em câncer de mama e que esta proteína pode ter um papel importante na morfogênese da glândula mamária. Além disso, a investigação em células MCF-7 demostrou que a expressão de Par-4 aumenta a sensibilidade das células ao tratamento com docetaxel. Com objetivo de entender melhor o papel de Par-4 em células tumorais de mama, nós investigamos o efeito da supressão de Par-4 nas células MDA-MB-231 in vitro e in vivo. A redução da expressão foi alcançada a partir de transfecção das células com vetores plasmidiais contendo shRNAs específicos para o transcrito de Par-4 e subsequentemente foram realizados ensaios funcionais. A expressão reduzida de Par-4 foi capaz de aumentar a capacidade de formação de colônias das células MDA-MB-231 em cultura. Além disso, as células MDA-MB-231 transfectadas com shRNA-Par-4 tiveram uma redução significativa da morte celular (fase sub-G0\\G1 do ciclo celular), em particular da morte por apoptose (Anexina-FITC/PI), após o tratamento com diferentes quimioterápicos. Em nosso estudo, as células MDA-MB-231-Controle tratadas com docetaxel apresentaram aumento nos níveis de Par-4 secretado, o que não foi observado nas células MDA-MB-231-shPar-4. Finalmente, nossos resultados in vivo sugerem que a expressão diminuída de Par-4 pode modular o crescimento tumoral em camundongos Balb/c NUDE. Em conjunto, nossos dados colaboram com o papel supressor de Par-4 já descrito na literatura e confirmam sua ação supressora em diferentes linhagens de câncer de mama / Breast cancer is the most incident tumor and the leading cause of mortality among women worldwide. Like other forms of cancer, breast cancer has a complex, heterogeneous and multifactorial etiology. The pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also known as PAR-4, Prostate Apoptosis Response-4) is known to selectively induce apoptosis in a wide variety of cancer cells. The role of Par- 4 as a tumor suppressor has been well established in recent years. However, little has been explored about the role and mechanisms of Par-4 suppressor function in breast cancer. Previous work from our research group demonstrated that the reduced expression of Par-4 is associated with a worse prognosis in breast cancer. Also an important role of Par-4 in the morphogenesis of the mammary gland was suggested. In addition, Par-4 overexpression in MCF-7 cells increased the sensitivity to docetaxel treatment. In order to better understand the role of Par-4 in breast tumor cells, we investigated the effect of Par-4 knock-down on MDA-MB-231 cells in vitro and in vivo. We performed shRNA-mediated Par-4 knockdown, and then carried out functional assays. The reduced expression of Par-4 was able to increase the colony formation capacity of MDA-MB-231 cells in culture. In addition, shRNA-Par-4 transfection in MDA-MB-231 cells led to a significant reduction of cell death (sub-G0/G1 cell cycle), particularly by apoptosis (Annexin-FITC/PI), after treatment with different chemotherapeutic agents. In our study, docetaxel-treated MDA-MB-231-Control cells showed increased levels of secreted Par-4, which was not observed in MDA-MB-231- shPar-4 cells. Finally, our in vivo results suggest that diminished Par-4 expression may modulate tumor growth in Balb/c NUDE mice. Taken together, our data support the suppressor role of Par-4 already described in the literature and confirm its suppressive action in different breast cancer cell lines

O câncer de mama é a neoplasia de maior ocorrência na população feminina. É uma doença hormônio-dependente com etiologia complexa e multifatorial. O câncer de mama invasivo é uma das principais causas da morbidade e mortalidade de mulheres no mundo. O desenvolvimento e a progressão de câncer de mama estão associados com acúmulo de várias alterações genéticas e epigenéticas, que resultam na expressão gênica diferencial entre células normais e tumorais. A hipóxia pode ser considerada uma das características principais de tumores sólidos e uma força motriz para a sobrevida das células tumorais e progressão maligna. O gene supressor de tumor PAWR (PKC apoptosis WT1 regulator; também denominado PAR-4, prostate apoptosis response-4) tem importante papel na apoptose tanto pela via intrínseca quanto pela via extrínseca e pode ser considerado um alvo para a terapia seletiva de células tumorais uma vez que a expressão de PAR-4 aumenta a sensibilidade da maioria das células de câncer a um segundo estímulo apoptótico. Alguns estudos, incluindo os de nosso grupo, têm mostrado o envolvimento de PAR-4 em câncer de mama e seu papel como fator prognóstico e preditivo de resposta a quimioterapia. No entanto, pouco é conhecido sobre o papel funcional deste gene ou os mecanismos envolvidos na regulação da expressão de PAR-4 na glândula mamária e no processo de tumorigênese da mama. No presente trabalho investigamos os efeitos da hipóxia na expressão de PAR-4 nas linhagens celulares MCF10A, MCF7 e MDA-MB-231. Para isto, as células foram incubadas por períodos de 30 minutos, 1, 2, 6 ou 24 horas em ambiente controlado com 5 % CO2, 95% N2 e 0.2% O2. A expressão de Par-4 foi avaliada quanto aos transcritos, por PCR em Tempo Real e quanto à expressão protéica, por western blot. Observamos que a baixa concentração de oxigênio diminui a expressão do mRNA de PAR-4, em tempos de 6 e 24 horas, nas linhagens MCF10A e MCF7 e no tempo de 24h nas células MDA-MB-231. Já quando os níveis protéicos de Par-4 foram analisados não foi observada diferença significativa nessas células após exposição à condição de hipóxia. A expressão do gene NDRG1, a qual se encontra aumentada em condições de hipóxia, foi utilizada como controle. Nossos dados sugerem que a expressão dos transcritos do gene supressor de tumor PAR-4 é inibida pelos mecanismos celulares de sobrevivência celular ativados durante a hipóxia / Breast cancer has the highest incidence rate of all malignant neoplasias affecting women. It is a hormone dependent disease with complex and multifactorial etiology. Invasive breast cancer is one of the principal causes of women morbidity and mortality in the world. Breast cancer development and progression are associated with accumulation of various genetic and epigenetic alterations, which result in differential gene expression among normal and tumor cells. Hypoxia could be considered one of the principal hallmarks of solid tumors and the driving force for the tumor cell survival and malignant progression. The tumor suppressor gene PAWR (PKC apoptosis WT1 regulator; also known as PAR-4, prostate apoptosis response-4) has an important role in apoptosis either by intrinsic or extrinsic pathways and could be considered as a target for selective therapy of tumor cells. Some studies, including of our group, have shown the PAR-4 involvement in breast cancer and its role as prognostic and predictive factor in response to chemotherapy. However, little is known about functional role of this gene or the mechanisms involved in PAR-4 expression regulation in mammary gland and in breast tumorigenesis process. In the present study we evaluated the hypoxia effects on PAR-4 expression in MCF10A, MCF7 and MDA-MB-231 cell lines. Therefore, the cells were incubated for time periods of 30 minutes, 1, 2, 6 or 24 hours under controlled environment with 5 % CO2, 95% N2 e 0.2% O2. PAR-4 expression was evaluated for transcripts, by Real Time PCR, and for protein expression, by western blot. We observed that low oxygen concentration decreases PAR-4 mRNA expression, in periods of 6 and 24 hours, in MCF10A and MCF7 cell lines and in the period of 24 hours in MDA-MB-231 cells. However, when the Par-4 protein levels were evaluated, no significant difference was observed in these cells after incubation under hypoxic conditions. NDRG1 gene expression, which is increased under hypoxia, was used as a control. Our data suggest that the expression of tumor suppressor gene PAR-4 transcripts is inhibited by cell survival mechanisms activated during hypoxia

Despite distinct dissimilarities, diverse cancers express several common pro-tumorigenic traits. We present here evidence that the pro-apoptotic protein Par-4 utilizes one such common tumorigenic trait to become selectively activated and induce apoptosis in cancer cells. Elevated PKA activity noted in cancer cells activated the apoptotic function of ectopic Par-4 or its SAC domain, which induces apoptosis selectively in cancer cells and not in normal or immortalized cells. PKA preferentially phosphorylated Par-4 at the T155 residue within the SAC domain in cancer cells. Moreover, pharmacological-, peptide- or siRNA-mediated inhibition of PKA activity in cancer cells resulted in abrogation of both T155 phosphorylation and apoptosis by Par-4. The mechanism of activation of endogenous Par-4 was similar to that of ectopic Par-4, and in response to exogenous stimuli, endogenous Par-4 induced apoptosis by a PKA and phospho-T155 dependent mechanism. Enforced elevation of PKA activity in normal cells resulted in apoptosis by the SAC domain of Par-4 in a T155-dependent manner. Together, these observations suggest that selective apoptosis of cancer cells by the SAC domain of Par-4 involves phosphorylation of T155 by PKA. These findings uncover a novel mechanism engaging PKA, a pro-cancerous activity commonly elevated in most tumor cells, to activate the cancer selective apoptotic action of Par-4.

Par‐4 is a pro‐apoptotic tumor suppressor that is mutated, suppressed or inactivated in cancer. Par‐4 exploits components of the extrinsic pathway to cause apoptosis selectively of cancer cells. This study identified Par‐4 as an essential component of the apoptotic pathway induced by TRAIL, which selectively targets cancer cells. RNA interference‐mediated knockdown of Par‐4 rendered cancer cells unresponsive to TRAIL‐induced apoptosis. Cells with knocked‐down levels of Par‐4 were deficient in the activation of the apoptosis‐initiator caspase‐8 and the apoptosis‐effector caspase‐3 in response to TRAIL. Par‐4 was identified as a critical mediator of membrane translocation of caspase‐8 and the adapter protein FADD. Surprisingly, Par‐4 was also found to interact with caspase 8 in untreated cells, and was cleaved at the N‐terminus at aspartic acid residue 123 in response to TRAIL. This, along with another cleavage by caspase‐9 effectively generated a fragment containing the functional module of Par‐4, the SAC domain, which is sufficient for apoptosis of cancer cells. Moreover, TRAIL activated caspase‐8 was also found to be involved in nuclear translocation of Par‐4, a crucial step during apoptosis induction by Par‐4. Together, our findings suggest that Par‐ 4 is an essential downstream target of caspase‐8 that is activated by TRAIL signaling and that, in turn, activates caspase‐8 and the downstream apoptotic pathway in response to TRAIL.

Chronic Lymphocytic Leukemia (CLL) is defined by the accumulation of clonally expanded CD5+ and CD19+ B lymphocytes in blood and secondary lymphoid organs with impaired apoptotic mechanisms. CLL represents one third of all leukemia cases with an average age of 72 years at diagnosis making it the most common adult leukemia. The Eµ-Tcl1 mouse serves as an excellent model to study the development of CLL as they progress to a CLL like disease by 9-14 months of age, due to overexpression of an oncogene, T cell Leukemia 1(Tcl1), specifically in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5+CD19+ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to the development of a CLL like disease within 3-8 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, spleen ultrasonography and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 protein (Par-4), a known pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers and has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. We found that CLL cells have constitutively active B-cell receptor signaling (BCR) and that inhibition of BCR signaling with FDA approved drugs causes a decrease in Par-4 protein, mRNA levels, and an increase in apoptosis. In particular, activities of Src family kinases, spleen tyrosine kinase and Bruton’s tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling in both Eµ-Tcl1 CLL cells and primary human CLL samples. Consistent with this, lenti-viral shRNA mediated knockdown of Lyn kinase leads to a decrease in Par-4 expression in MEC-1 cells, a human CLL derived cell line. Igα (CD79a) silencing in primary human CLL cells also results in down regulation of Par-4 expression. Additionally, we knocked down expression of Par-4 in MEC-1 cells which resulted in a decrease in cell growth that could be attributed to an increase in p21 expression and a reduction in the G1/S cell cycle transition. We have also observed this phenomenon by crossing mice deficient in Par-4 with the Eµ-Tcl1 mouse where lack of Par-4 delays CLL growth in the mouse significantly (time to euthanization due to poor body condition - Eµ-Tcl1: 8.9mo vs Par4-/-EµTcl1: 11.97 mo, p = 0.0472) and splenic B-CLL cells from these mice also have increased expression of p21. Since mice in this cohort are whole body knockout for Par-4, the difference in survival times between the Par-4 +ve and Par-4 –ve EµTcl1 mice could be due to the influence of Par-4 on CLL cells as well as the effect of Par-4 secreted by the CLL cells on the microenvironment. There could be other potential roles for Par-4 in the context of CLL which are under further investigation. We have also investigated the site of CLL growth in mouse models to determine that the spleen is the primary organ to accumulate the CLL tumor burden. We have found that splenectomy significantly delays the development of CLL in the primary Eμ-Tcl1 mouse model and prevents growth and development in the adoptive transfer model. Interestingly, splenectomy did not delay CLL development as significantly in animals deficient for Par-4 compared to C57BL/6 wild type mice. Par-4 appears to regulate a specific microenvironment required for CLL growth. Current studies are investigating the role of Par-4 in the microenvironment and the cell types that are critical for CLL growth within the splenic niche.