Charusheila Ramkumar

Regulation of Senescence in Cancer and Aging

Senescence is regarded as a physiological response of cells to stress, including telomere dysfunction, aberrant oncogenic activation, DNA damage, and oxidative stress. This stress response has an antagonistically pleiotropic effect to organisms: beneficial as a tumor suppressor, but detrimental by contributing to aging. The emergence of senescence as an effective tumor suppression mechanism is highlighted by recent demonstration that senescence prevents proliferation of cells at risk of neoplastic transformation. Consequently, induction of senescence is recognized as a potential treatment of cancer. Substantial evidence also suggests that senescence plays an important role in aging, particularly in aging of stem cells. In this paper, we will discuss the molecular regulation of senescence its role in cancer and aging. The potential utility of senescence in cancer therapeutics will also be discussed.

Smurf2 suppresses B-cell proliferation and lymphomagenesis by mediating ubiquitination and degradation of YY1

About half of patients with diffuse large B-cell lymphoma (DLBCL) do not respond to or relapse soon after the standard chemotherapy, indicating a critical need to better understand the specific pathways perturbed in DLBCL for developing effective therapeutic approaches. Mice deficient in the E3 ubiquitin ligase Smurf2 spontaneously develop B-cell lymphomas that resemble human DLBCL with molecular features of germinal center or post-germinal center B cells. Here we show that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal center transcription factor. Smurf2 deficiency enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, Smurf2 expression is significantly decreased in primary human DLBCL samples, and low levels of Smurf2 expression correlate with inferior survival in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, suggesting pharmaceutical targeting of Smurf2 as a new therapeutic paradigm for DLBCL.

Smurf2 regulates hematopoietic stem cell self-renewal and aging.

The age‐dependent decline in the self‐renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age‐dependent decline of stem cell self‐renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2‐deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2‐deficient mice had a significantly better repopulating capacity than aged wild‐type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2‐deficient HSCs exhibited elevated long‐term self‐renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self‐renewal and aging.

A Circulating microRNA Signature Predicts Age-Based Development of Lymphoma.

Extensive epidemiological data have demonstrated an exponential rise in the incidence of non-Hodgkin lymphoma (NHL) that is associated with increasing age. The molecular etiology of this remains largely unknown, which impacts the effectiveness of treatment for patients. We proposed that age-dependent circulating microRNA (miRNA) signatures in the host influence diffuse large B cell lymphoma (DLBCL) development. Our objective was to examine tumor development in an age-based DLBCL system using an inventive systems biology approach. We harnessed a novel murine model of spontaneous DLBCL initiation (Smurf2-deficient) at two age groups: 3 and 15 months old. All Smurf2-deficient mice develop visible DLBCL tumor starting at 15 months of age. Total miRNA was isolated from serum, bone marrow and spleen and were collected for all age groups for Smurf2-deficient mice and age-matched wild-type C57BL/6 mice. Using systems biology techniques, we identified a list of 10 circulating miRNAs being regulated in both the spleen and bone marrow that were present in DLBCL forming mice starting at 3 months of age that were not present in the control mice. Furthermore, this miRNA signature was found to occur circulating in the blood and it strongly impacted JUN and MYC oncogenic signaling. In addition, quantification of the miRNA signature was performed via Droplet Digital PCR technology. It was discovered that a key miRNA signature circulates throughout a host prior to the formation of a tumor starting at 3 months old, which becomes further modulated by age and yielded calculation of a ‘carcinogenic risk score’. This novel age-based circulating miRNA signature may potentially be leveraged as a DLBCL risk profile at a young age to predict future lymphoma development or disease progression as well as for potential innovative miRNA-based targeted therapeutic strategies in lymphoma.

Serial transplantation of bone marrow to test self-renewal capacity of hematopoietic stem cells in vivo.

Hematopoietic stem cells (HSCs) have the ability to self-renew and replenish the blood and immune system for the life span of an individual. An age-associated decline in HSC function is responsible for the decreased immune function and increased incidence of myeloid diseases and anemia in the elderly. The changes in HSC function are thought to occur as the result of an intrinsic defect in the self-renewal potential of HSCs as they age. In this chapter, we describe a bone marrow serial transplantation protocol designed to test the self-renewal capacity of HSCs in vivo.

Abstract 1994: Smurf2 mediates ubiquitination and degradation of YY1 to suppress B-cell proliferation and lymphomagenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Current treatment of diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkins lymphoma, has improved patient survival significantly. However, 40-50% of DLBCL patients still die from this disease. There is a critical need to better understand the specific molecular pathways that are perturbed in DLBCL for the development of new and effective therapeutic approaches. We show that mice deficient in the E3 ubiquitin ligase Smurf2 spontaneously develop B-cell lymphomas that resemble human DLBCL with molecular features of germinal center B cells. We discover that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal center transcription factor. Importantly, Smurf2 deficiency enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, the expression of Smurf2 is significantly decreased in primary human DLBCL samples compared to normal B cells, and low levels of Smurf2 expression correlate with poor survival prognosis in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, suggesting Smurf2 as a new therapeutic paradigm for DLBCL. Citation Format: Charusheila Ramkumar, Hang Cui, Yahui Kong, Stephen Jones, Rachel Gerstein, Hong Zhang. Smurf2 mediates ubiquitination and degradation of YY1 to suppress B-cell proliferation and lymphomagenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1994. doi:10.1158/1538-7445.AM2013-1994 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2914: Mice deficient in Smurf2 exhibit increased spontaneous tumorigenesis

Smurf2, a HECT family E3 ubiquitin ligase has been shown to ubiquitinate and target for degradation of several proteins that promote tumorigenesis, including TGF-β receptors, Smad2/3, β-catenin, and Mad2. Furthermore, we have previously found that Smurf2 can induce senescence in early passage cells in culture. As senescence has been postulated to act as a tumor suppression mechanism, we hypothesize that Smurf2 functions as a tumor suppressor. To test this hypothesis, we generated a Smurf2-deficienct mouse strain using a gene trapping strategy, in which transcription of Smurf2 is compromised. The homozygous Smurf2-deficient mice were obtained with the expected Mendelian ratio after intercrossing of heterozygous mice, and they exhibited no obvious developmental defects. Analysis of mouse embryonic fibroblasts from these Smurf2-deficient mice revealed that they exhibited delayed senescence entry and consequently had an increased tendency to become immortalized in culture. Cohorts of Smurf2-deficient and wild-type mice were generated and monitored for spontaneous tumor development. In this ongoing study, 37% (13 out of 35) of the Smurf2-deficient mice, but none of the wild-type mice showed signs of morbidity after 14-month of age. The moribund Smurf2-deficient mice were found to have enlarged spleen, pale kidneys and liver, and/or enlarged lymph nodes. Histopathology analysis indicated that most of the tumors are lymphomas. In addition, adenocarcinoma of the small intestine was found in one Smurf2-deficient mouse. Collectively, our data suggest that Smurf2 is a tumor suppressor. Preliminary studies of the underlying molecular mechanism of tumorigenesis in Smurf2-deficient mice will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2914.