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Why study T-cell leukemia?

While many cancer patients have benefited from the wonders of targeted agents and immunotherapies, patients with the T-cell subtype of acute lymphoblastic leukemia (T-ALL) are falling behind. The primary treatment remains chemotherapy and radiation. Acute lymphoblastic leukemia is the most common childhood cancer and is rising in incidence. T-ALL also affects adults where cure rates are abysmal at ~35%.  Even if cure is achieved, the battle is not over for children and adults. Survivors of ALL must fight serious toxicities for the rest of their lives, like early dementia, learning disabilities, seizures, psychiatric illnesses, stroke, osteoporosis, heart disease, nerve damage, and second cancers. There is an urgent need for effective and safer treatments for ALL.

Why study T-cell regeneration?

Infection due to prolonged T-cell deficiency after chemotherapy or bone marrow transplantation (BMT) remains a major clinical problem. For this reason, interventions that stimulate T-cell developmental pathways to regenerate the T-lineage and restore T-cell immunity are being actively studied. However, these strategies can be problematic as supraphysiological activation of some pathways can lead to excessive T-cell commitment and depletion of stem cells. In contrast, supraphysiological activation of other pathays can preserve stem cells and promote proliferation without inducing leukemia. By studying leukemia and early T-cell progenitor biology in parallel, we learn how to activate  cellular pathways in ways that could be used to accelerate thymic regeneration after cancer therapy to restore the immune system's ability to fight infection.

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The critical questions we face

How do transcription factor oncogenes "hijack" pro-proliferative pathways of normal T cell precursors to convert them into leukemia stem cells? Can these pathways be experimentally amplified to promote T-cell regeneration without causing cancer? What are the critical signaling pathways and enhancer elements that leukemia cells and T-cell progenitors depend on? Can we disrupt protein-protein interactions of transcriptional cofactors/factors either genetically or with small compounds to target leukemia stem cells or restore T-cell immunity after irradiation or chemotherapy? To answer these questions, we use a multidisciplinary team-based approach integrating diverse cutting edge approaches with genetically engineered mouse models, patient-derived xenografts, transcriptional genomics, proteomics, and bioinformatics to investigate blood cell development and leukemogenesis.

The Notch pathway

NOTCH1 is the most prevalent oncogene in T-ALL. Unfortunately, in clinical trials, anti-Notch drugs had too much toxicity. They hurt the intestine, causing diarrhea. They hurt the skin, causing skin cancers. Doctors could only use low, ineffective doses of these drugs or had to stop the trial altogether. The reason for the toxicity is that Notch is very important for keeping normal tissues healthy. Notch activation is also the primary driver of T-cell commitment. Studies that activate Notch to restore T-cell immunity have had limited success in part due to depletion of stem cells.

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How can we knock out Notch in cancer, but preserve Notch in normal cells? How can we activate the Notch pathway to promote thymic regeneration but avoid depleting stem cells? We believe that the answer may be partner proteins called cofactors that stick to Notch.  These cofactors act through context-dependent mechanisms to modulate Notch activity. Our theory is that manipulating certain cofactors could combat Notch in cancer without intolerable side effects and restore T-cell immunity after cytoreductive therapies without depleting stem and progenitor cells.

Our vision

With the generous support from donors, foundations, the University of Michigan, and the NIH, we have published multiple reports supporting our theory (McCarter et al., Blood Cancer Discovery, 2020; Wang et al., Blood, 2018; Pinnell et al., Immunity, 2015). We showed that the cofactors ETS1 and ZMIZ1 stick to Notch and triggers its cancer-causing functions but not its normal functions. When Ets1or Zmiz1are blocked, leukemic mice live longer and are relatively healthy. Unlike Notch-deficient mice, Ets1-deficient or Zmiz1-deficient mice have relatively healthy intestine and skin. Disrupting the Zmiz1-Notch1 interaction impairs Notch complex recruitment to response elements and Myc transcription. Conversely, overexpressing Zmiz1 restored T-cell progenitor compartments without inducing leukemia.

 

This biochemical separation of Notch functions stems from an evolutionary conserved principle of the tissue-restricting and amplifying role of Notch cofactors, already established in fruit flies. This principle might be exploited to disable Notch-dependent gene expression programs in cancer with less toxicity than pan-Notch inhibitors. It is possible to disengage activated Notch from its chromatin functions in cancer cells without directly targeting Notch complex formation. As a physician who actively takes care of leukemia patients, I see firsthand how safe targeted drugs are urgently needed for T-ALL patients. If drugs are like scalpels, our work provides the knowledge of anatomy to guide the surgical oncologist’s blade to excise cancer without damaging healthy tissues.

Mentorship

We are concerned about the critical need to carefully train the next generation of scientists and leaders in biomedical research in academia and industry as well as alternative careers. Trainees will be working in a direct mentor-mentee relationship. Training will be tailored according the individual needs and level. Examples include personalized lectures; teaching on how to critically assimilate the literature; teaching on how to generate focused, clinically relevant questions/hypotheses; teaching on how to test hypotheses with well-designed experiments; and teaching on basic/advanced laboratory techniques.

 

Project 1

The trainee will determine significance and mechanisms by which a collaborative Notch cofactor co-binds and co-regulates oncogenic Notch target genes in large transcriptional complexes sitting at Notch-responsive super-enhancers. We wil knockout this cofactor in mice to assess its role in T-cell development and leukemogensis; as well as assess potential toxicities of systemic inhibition. We will map cofactor interactions  at the amino acid level. We will identify ways to disrupt these interactions to downregulate oncogene expression and impair tumor growth in vitro and in vivo.

 

Project 2

The trainee will determine the significance and mechanism of a previously unrecognized super-enhancer where teams of transcriptional players join forces with Notch to collectively activate oncogenic transcriptional effector pathways in a tissue-specific manner. We will knock out this enhancer in mice to understand its role in Notch-induced T-cell progenitor self-renewal and leukemogenesis; as well as assess potential systemic toxicities of inactivating this enhancer. We will identify the transacting factors that bind this enhancer and inactivate this enhancer in human patient-derived leukemia cells

Project 3

The trainee will determine the significance and mechanism of leukemia-associated transcription factors and epigenetic modifers in thymic stem cell development and regeneration after irradiation or chemotherapy. The trainee will use conditional knockout mice to map the hematopoietic defects in vitro and in vivo. We will use transcriptional genomic and proteomic technologies to identify the function and mechanism of the transcription factor and enhancer networks of target genes and effector pathways that drive self-renewal of rare progenitor stem cells from which leukemia originates.

Methodologies

  • High-speed multicolor flow cytometric acquisition and sorting

  • Histopathological analysis

  • Next-generation sequencing (ChIP-Seq, RNA-Seq, ATAC-Seq)

  • Bioinformatic/biostatistical analyses

  • CRISPR/lentiviral-based genetic modification of leukemic cells

  • Chromatin immunoprecipitation

  • Mass spectrometry

  • Nuclear magnetic resonance

  • Robotic  screening of chemical and RNAi libraries

Model systems of hematopoiesis and leukemia

  • In vitro model systems of the lymphopoiesis and T-ALL proliferation

  • Retroviral murine bone marrow transplantation

  • Patient-drived leukemia xenograft models

  • Conditional knockout mice

  • Inducible fluorescent mouse models

  • Leukemia stem cell assays

Selected publications

Chiang, M.Y., Xu, L., Histen, G., Shestova, O., Roy, M., Nam, Y., Blacklow, S.C., Sacks, D., Pear, W.S., Aster, J.C.: Identification of a conserved negative regulatory sequence that influences the leukemogenic activity of NOTCH1. Mol. Cell. Bio. 16:6261-6271, 2006.

Chiang, M.Y., Xu, L., Shestova, O., Histen, G., L’Heureux, S., Romany, C., Childs, M.E., Gimotty, P.A., Aster, J.C., Pear, W.S.: Leukemia-associated NOTCH mutations are weak tumor initiators, but accelerate K-ras-initiated leukemia. J. Clin. Invest. 118:3181-3194, 2008.

Yashiro-Ohtani, Y., He Y., Ohtani, T., Jones, M.E., Shestova, O., Xu, L., Fang, T.C., Chiang, M.Y., Intlekofer, A.M., Zhuang, Y., Pear, W.S.: Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A signaling at the beta-selection checkpoint. Genes and Dev. 23:1665-76, 2009.

Liu, H., Chi, A.W.S., Chiang, M.Y., Arnett, K.L., Xu, L., Shestova, O., Wang, H., Li, Y., Bhandoola, A., Aster, J.C., Blacklow, S. Pear, W.S. Notch dimerization is required for leukemogenesis and T cell development. Genes and Dev. 21:2395-407, 2010

Ashworth, T.D., Pear, W.S., Chiang, M.Y., Blacklow, S.C., Mastio, J., Xu, L., Kelliher, M., Kastner, P., Chan, S., Aster, J.C. Deletion-based mechanisms of Notch1 activation in T-ALL: Key roles for RAG recombinase and a conserved internal translational start site in Notch1. Blood. 116:5455-64, 2010

Rakowski, L.A., Lehotzky, E.A., Chiang.M.Y. Transient Responses to NOTCH and TLX1/HOX11 Inhibition in T-cell Acute Lymphoblastic Leukemia/Lymphoma. PLoS ONE. 6:e16761, 2011.

Liu, H., Pear, W.S., Chiang, M.Y.* Critical Roles of NOTCH1 in Acute T-cell Lymphoblastic Leukemia/Lymphoma. Int. J. of Hematology. 2:118-25, 2011.

Kiel, M.J., Velusamy, T., Betz, B.L., Zhao, L., Weigellin, H.G., Chiang, M.Y., Huebner-Chan, D.R., Bailey, N.G., Yang, D.T., Bhagat, G., Miranda, R.N., Bahler, D.W., Medeiros, L.J., Lim, M.S., Elenitoba-Johnson, K.S.J. Whole genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma. Journal of Experimental Medicine. 209: 1553-1551, 2012.

Giambra, V., Jenkins, C.R., Wang, H., Lam, S., Shevchuk, O., Nemirovsky, O., Wai, C., Gusscott, S., Chiang, M.Y., Aster, J.C., Humphries, R.K., Eaves, C., Weng, A.P. PKCθ Regulates T-Cell Leukemia-Initiating Activity via Reactive Oxygen Species. Nature Medicine. Nature Medicine. 2012. Epub ahead of print. PMID: 23086478

Chiang, M.Y.*, Shestova, O., Xu, L., Aster, J.C., Pear, W.S. Divergent effects of supraphysiological Notch signals on leukemia stem cells and hematopoietic stem cells. Blood. 121: 905-17, 2013. PMID: 23115273.

Rakowski, L.A., Garagiola, D.G., Li, C.M., Decker, M., Caruso, S., Jones, M., Kuick, R., Cierpicki, T., Maillard, I., and Chiang, M.Y. Convergence of the ZMIZ1 and NOTCH1 pathways at C-MYC in acute T lymphoblastic leukemias. Cancer Research. 73: 930-41, 2013. PMID: 23161489. PMCID: PMC3549029.

Pinnell NE and Chiang MY. Collaborating Pathways that Functionally Amplify NOTCH1 Signals in T-Cell Acute Lymphoblastic Leukemia. J Hematol Transfus. 2013. 1: 1004.

 

Lubeck BA, Lapinski PE, Oliver JA, Ksionda O, Parada LF, Zhu Y, Maillard I, Chiang MY, Roose J, and King PD. Co-deletion of the Ras GTPase-activating proteins Neurofibromin 1 (NF1) and p120 RasGAP (RASA1) in T cells results in the development of T cell acute lymphoblastic leukemia. J Immunol. 2015. 195:31-5. PMID: 26002977.

 

Pinnell N, Yan R, Cho H, Keeley T, Murai M, Liu Y, Serna Alarcon A, Qin, J., Wang, Q., Kuick R, Elenitoba-Johnson KSJ, Maillard I, Samuelson LC, Cierpicki T, Chiang MY. The PIAS-like coactivator Zmiz1 is a direct and selective cofactor of Notch1 in T-cell development and leukemia. Immunity. 43: 1-14. 2015.

 

Chiang MY, Radojcic V, Maillard, I. Oncogenic Notch signaling in T and B cell lymphoproliferative disorders. Curr Opin Hematol. 2016. PMID: 27135981. PMCID: PMC496255.

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Chiang MY*, Wang Q, Gormley AC, Stein, SJ, Xu L, Shestova O, Aster JC, Pear WS. High selective pressure for Notch1 mutations that induce Myc in T cell acute lymphoblastic leukemia. Blood. 128: 2229-2240. 2016. PMID 27970423.

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Gormley A, Wang Q, Chiang MY. Notch in Leukemia. Adv Exp Med Biol. 1066:355-394. 2018. PMID 30030836.

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 Wang, Q, Yan, R, Pinnell, N, Gormley, A, Oh, Y, Liu, Y, Sha, C, Garber, NF, Chen, Y, Wu, Q, Ku, C, Tran, I, Serna Alarcon, A, Kuick, R, Engel, JD, Maillard, I, Cierpicki, T, and Chiang, MY.  Stage-specific roles for Zmiz1 in Notch-dependent steps of early T-cell development. Blood. 132(12):1279-1292. 2018. PMID 30076146

 

Chiang MY. Finding ARIEL under the sea of T-ALL circuits. Blood. 2019 Jul 18;134(3):219-221. doi: 10.1182/blood.2019001584. PubMed PMID: 31320362; PubMed Central PMCID: PMC6639983.

 

Rodriguez S, Abundis C, Boccalatte F, Mehrotra P, Chiang MY, Yui MA, Wang L, Zhang H, Zollman A, Bonfim-Silva R, Kloetgen A, Palmer J, Sandusky G, Wunderlich M, Kaplan MH, Mulloy JC, Marcucci G, Aifantis I, Cardoso AA, Carlesso N. Therapeutic targeting of the E3 ubiquitin ligase SKP2 in T-ALL. Leukemia. 2020 May;34(5):1241-1252. doi: 10.1038/s41375-019-0653-z. Epub 2019 Nov 26. PubMed PMID: 31772299; PubMed Central PMCID: PMC7192844.

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Chiang MY. Tearing ATL apart to find HTLV's sinister plans. Blood. 2020 Mar 19;135(12):887-889. doi: 10.1182/blood.2020004998. PubMed PMID: 32191800.

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McCarter, A.M., Della Gatta, G., Melnick, A., Kim, E., Sha, C., Wang, Q., Nalamolu, J.K., Liu, Y., Keeley, T.M. Yan, R., Sun, M., Kodgule, R., Kunnath, N., Ambesi-Impiombato, A., Kuick, R., Rao, A., Ryan, R.J.H., Kee, B.L., Samuelson, L.C., Ostrowski, M.C., Ferrando, A.A., and Chiang, M.Y. Combinatorial ETS1-dependent control of oncogenic NOTCH1 enhancers in T-cell leukemia. Blood Cancer Discovery. Published OnlineFirst on July 14, 2020. 

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Chiang MY. Firing up chromatin to forge T-ALL. Blood. 2022 Apr 21;139(16):2418-2420. doi: 10.1182/blood.2021015364. PubMed PMID: 35446377; PubMed Central PMCID: PMC9029094.

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Kodgule R, Goldman JW, Monovich AC, Saari T, Aguilar A, Hall CN, Rajesh N, Gupta J, Chu SA, Yi L, Gurumurthy A, Iyer A, Brown NA, Chiang MY, Cieslik MP, Ryan RJH. ETV6 Deficiency Unlocks ERG-Dependent Microsatellite Enhancers to Drive Aberrant Gene Activation in B-Lymphoblastic Leukemia. Blood Cancer Discov. 2023. 2023 Jan 6;4(1):34-53. doi: 10.1158/2643-3230.BCD-21-0224. PMID: 36350827 PMCID: PMC9820540

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Melnick, A.F., Mullin, C., Lin, K., McCarter, A.C., Liang, S, Liu, Y., Wang, Q., Dean, N.A., Choe, E., Kunnath, N, Bodanapu, G, Akter, F, Magnuson, B, Kumar, S, Lombard, D.B., Muntean, A.G Ljungman, M., Sekiguchi, J., Ryan, R.J.H., and Chiang, MY. Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress. Blood. 2023. (In press).

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