Cellular growth (size increase) is a fundamental biological process that is required for subsequent cell division in most cases. The adaptive immune response of vertebrates occurs through clonal selection where a single lymphocyte with a randomly generated receptor undergoes rapid proliferation to generate many more cells with the same receptor. Given the potential of an invading pathogen to replicate rapidly, the cell cycle time of lymphocytes upon activation is uniquely short where division happens every four to six hours. Prior to this proliferative burst however, there is a long phase of cell growth (blastogenesis) that persists for about 24-36 hours. The ultimate goal of my research is to investigate lymphocyte blastogenesis at the molecular level using chemical and genetic approaches.
The mammalian target of rapamycin (mTOR) pathway is a bona fide master regulator of cell growth in many different organisms and cell types. mTOR is a serine/threonine kinase that promotes cell growth by upregulating anabolic processes such as protein synthesis, lipid biogenesis, and nucleotide synthesis. Interestingly, rapamycin (RAP), a highly selective chemical inhibitor of mTOR, exhibits potent anti-proliferative activity against lymphocytes and received FDA approval for the prevention of organ rejection more than 15 years ago. Surprisingly, the precise mechanism of action of RAP in lymphocytes is largely unknown. My interest in cell growth control in lymphocytes and the unclear mechanism of RAP as a therapeutic agent led me to investigate the downstream targets of mTOR to potentially discover novel effectors of mTOR required for lymphocyte blastogenesis.
The lab has generated a novel mouse strain where mTOR kinase activity can be abolished specifically in mature T cells (mTOR-KI; kinase-inactive). mTOR-KI T cells phenocopies the growth and division defect seen in complete mTOR knockout (mTOR-KO) T cells and also T cells that lack a critical component of mTOR complex 1 (mTORC1), raptor. This suggests that lymphocyte growth and division requires the kinase activity of mTORC1.
Two of the most studied direct mTORC1 kinase substrates are the ribosomal protein S6 kinases (S6Ks) and the translation initiation factor 4e-binding proteins (4E-BPs). S6Ks have been implicated in cell growth control in many different contexts. For example, flies and mice that lack S6K are smaller in size due to a decrease in cell size. Interestingly, fibroblasts that lack S6Ks are also smaller in size but show normal cell division rates. 4E-BPs are negative regulators of the cap-binding protein eIF4e. Phosphorylation of 4E-BPs by mTOR releases this negative regulation thereby promoting cap-dependent translation. In fibroblasts, the 4E-BP/eIF4e axis downstream of mTOR has been shown to mainly regulate cell division and not cell size by selective translation of key cell cycle regulators.
We are investigting the role of these two effectors in lymphocytes using chemical and genetic approaches. To investigate the role of S6Ks in lymphocytes, we will use several approaches. First approach will involve the use of lymphocytes that lack S6Ks (S6K1 and S6K2). Since these mice exhibit perinatal lethality, to overcome the potential developmental compensatory effects, we will utilize a novel S6K1 specific inhibitor in lymphocytes that lack S6K2. This chemical genetic approach will overcome chronic genetic deletion of S6Ks by acutely inhibiting all S6K activity.
To investigate the role of 4E-BPs, we will use a system to overexpress a mutant form of 4E-BP1 that cannot be phosphorylated by mTOR (five phosphorylation sites are mutated to alanine; 4E-BP1-5A). Overexpression of 4E-BP1-5A can be induced in a doxycycline-dependent manner in our system and we will investigate two questions. One is whether 4E-BP1-5A expression prior to activation is critical for lymphocyte growth and subsequent division. Second is the expression of 4E-BP1-5A once the lymphocytes have entered cell cycle after the blastogenesis phase. These studies will hopefully illuminate novel effectors of mTORC1 in lymphocytes and potentially lead to more selective targeting of the mTOR pathway to modulate immunity.
Limon, J.L., So, L., Jellbauer, S., Chiu, H., Corado, J., Sykes, S., Raffatellu, M., and Fruman, D.A. (2014) mTOR kinase inhibitors promote antibody class switching. Submitted.
Yea, S.S., So, L., Mallya, S., Lee, J., Rajasekaran, K., Malarkannan, S., and Fruman, D.A. (2014) Effects of novel isoform-selective phosphoinositide 3-kinase inhibitors on natural killer cell function. PLOS ONE, in press.
Mallya, S., Fitch, B., Lee, J.S., So, L., Janes, M.R., and Fruman, D.A. (2013) Resistance to mTOR kinase inhibitors in a lymphoma cell line lacking 4EBP1. Plos One. 9(2):e88865
Roy, S., Stevens, M., So, L., and Edinger, A.L. (2013) Reciprocal effects of Rab7 deletion in activated and neglected T cells. Autophagy. 9(7): 1009-1023.
So, L., Yea, S.S., Oak, J.S., Manmadhan, A., Ke, Q.H., Janes, M.R., Li, L., Kessler, L., Kucharski, J., Martin, M., Ren, P., Jessen, K., Liu, Y., Rommel, C., and Fruman, D.A. (2013) Selective inhibition of phosphoinositide 3-kinase p110a preserves lymphocyte function. J. Biol. Chem. 288(8): 5718-5731.
So, L., and Fruman D.A. (2013) Phosphoinositide 3-kinase. Encyclopedia of Signaling Molecules. Springer, Inc. New York. 1392-1400.
So, L., and Fruman D.A. (2012) PI3K signaling in B- and T-lymphocytes: new developments and therapeutic advances. Biochem J. 442: 465-481.
Gokhale, A., Larimore, J., Werner, E., So, L., Moreno-De-Luca, A., Lese-Martin, C., Lupashin, V.V., Smith, Y., and Faundez, V. (2012) Quantitative proteomic and genetic analyses of the schizophrenia susceptibility factor dysbindin identify novel roles of the biogenesis of lysosome-related organelles complex 1. J. Neurosci. 32: 3697-3711.
Janes, M.R., Limon, J.J., So, L., Chen, J., Lim, R.J., Chavez, M.A., Vu, C., Lilly, M.B., Mallya, S., Ong, S.T., Konopleva, M., Martin, M.B., Ren, P., Liu, Y., Rommel, C., and Fruman, D.A. (2010) Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor. Nat. Med. 16: 205-213.
Honors and Awards:
1. NIH NIAID T32 Immunology Research Training Program (2013-2014)
2. William D. Redfield Graduate Fellowship for excellence in research (2013)
3. Honorable Mention for Best Talk at the Department of Molecular Biology and Biochemistry Retreat (2013)
4. IRIC Symposium Invited Student Talk in Montreal, CA (2012)