The proto-oncogene TCL1 is causally implicated in various B-cell and T-cell malignancies. Here, we aim to refine an integrative model of leukemogenic TCL1 acting on interlinked molecular axes such as antigen receptor stimulation, DNA damage response, and cell cycle control. Through a functional characterization of TCL1-engaged signaling networks and TCL1-centric therapeutic vulnerabilities, we addressed an essential scientific knowledge gap on the mechanisms of (chemo)therapy resistance in hematologic tumors.
Aberrantly high TCL1 levels are a hallmark of the lymphatic neoplasms chronic lymphocytic leukemia (CLL) and T-cell prolymphocytic leukemia (T-PLL), with highest levels signifying poorer clinical outcomes. TCL1 transgenic (tg) mice closely resemble human leukemogenesis. However, the oncogenic mechanisms of TCL1 are not fully understood. TCL1 shows no intrinsic enzymatic activity and has no DNA-binding motif. Several studies describe TCL1 being engaged into protein-protein interactions, yet, the functional consequences of such interplay are mainly unknown. Therefore, we comprehensively characterized the TCL1 interactome and the impact on TCL1-mediated signaling pathways on leukemogenesis.
In our previous work, we established TCL1 as a ‘sensitizer’ to T- and B-cell receptor signals via augmentation of AKT kinase activity. Here we show that TCL1 overexpression additionally dysregulates the DNA-damage response (DDR) and cell cycle regulation. We propose that the impact of TCL1 on cell signaling homeostasis (i. e. antigen-mediated responses, DNA repair, and cell cycle check-point transition) might assume a causative role in genomic instability and initiation / progression of leukemia (Fig. 1).
TCL1 has been previously shown to act as a chaper-on-like protein. To discover novel interacting partners of TCL1, we performed a mass spectrometry-based screen of TCL1-interacting proteins in CLL-derived B-cells (JVM3) and in primary CLL samples. In JVM3-TCL1A cells exposed to different conditions (unstimulated, genotoxic stress, and mitosis), we identified 1040 annotated proteins to interact with TCL1. Gene-set overrepresentation analysis revealed G2/M mitotic checkpoint transition and DDR as functional categories enriched in the TCL1 interactome. This was further confirmed by first-neighborhood analysis that defined G2/M mitotic checkpoint transition as one of the main TCL1-centric signaling nodes (copy-right regulations do not allow inclusion of primary data here).
We see that TCL1 co-localizes with the mitotic checkpoint protein CDC20 and its negative regulator MAD2 at the mitotic spindles during mitosis. The protein-protein interaction of TCL1 with CDC20 was validated in several models including B-cell lines as well as primary murine and human CLL B-cells. We further showed that complexing of CDC20 with TCL1 impacts the function of CDC20, a key player during G2/M cell cycle transition. Moreover, TCL1-tg cells showed reduced CDC20 protein expression, an accelerated cell cycle transition, and a significantly higher number of cells with multipolar spindles, suggesting an important role of TCL1 in mitotic transition. By using CDC20 mutant constructs and in silico modeling algorithm of protein-protein interactions HADDOCK2.2, we established a model of the TCL1-CDC20-MAD2 protein complex. Together, under the impact of TCL1, abrogated cell cycle transi-tion and aberrant formation of mitotic spindles in dividing B-cells lead to an increased accumulation of DNA lesions and genomic instability.
We also identified that TCL1 interacts with several members of the DDR. Two key molecules in sensing and processing of DNA lesions as well as in a proper cellular response are ATM and p53. Both were shown to interact with TCL1. Their phosphorylation/activity was reduced in TCL1-overexpressing cells and in Eµ-TCL1 B-cells. Consistently, a reduced expression of p53 target genes was found in splenocytes from Eµ-TCL1 mice. In line with this, leukemic TCL1-tg cells showed an enhanced accumulation of DNA double strand breaks. Furthermore, the rate of chromosomal gains or losses was significantly increased in TCL1-tg conditions compared to wild-type cells (Fig. 2). Together, TCL1 overexpression impairs the proficiency of the DDR by suppressing the ATM and p53 responses and by that conceivable promotes leukemogenesis.
In conclusion, our data indicate that TCL1 controls cellular responses not only via BCR/TCR induced pro-survival effectors like AKT, but also through other functional branches such as a DDR and mitotic transition control, indicating that multiple signaling events account for the overall oncogenic potential of TCL1.
We aim to characterize in depth the repressive impact of TCL1 on the activity and proficiency of ATM and p53. By using quantitative proteomics, we will identify TCL1-mediated changes in the constitutional make-up of p53 and ATM protein complexes upon DNA damage induction. Upregulation of TCL1 has been previously assigned to chemotherapy resistance in CLL and T-PLL. The underlying molecular mechanism of such resistance is however still elusive. Our data on a TCL1-mediated repression of ATM/p53 responses and a promotion of cell cycle checkpoint abrogation provide new insights on how TCL1 might mediate chemo-resistance in leukemias with intact ATM and TP53 genes, and might help in refining a treatment strategy for such patients. By molecular model-building of the impacts of TCL1 on the DDR and on cell cycle regulation, we will fill functional knowledge gaps and reveal TCL1-centric susceptibilities in TCL1-driven lymphoid neoplasms.
1. Schrader A., …, Vasyutina E., …, Herling M. (2018). Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL. Nat Commun 9(1), 697.
2. Andersson E.I., … Herling M., Mustjoki S. (2017) Dis-covery of novel drug sensitivities in T-PLL by high-throughput ex vivo drug testing and mutation profil-ing. Leukemia 32(3), 774-787.
3. Reiners K.S., …, Vasyutina E., …, Herling M. (2016). Extracellular vesicles released from chronic lympho-cytic leukemia cells exhibit a disease relevant mRNA signature and transfer mRNAs to by-stander cells. Haematologica 102(3), 100-103.
4. Lohmann G., Vasyutina E., …, Herling M. (2016). Tar-geting transcription-coupled nucleotide excision repair overcomes resistance in chronic lymphocytic leuke-mia. Leukemia 31(5), 1177-1186.
5. Lilienthal N., … Herling M. (2016). A Novel Recombi-nant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells. Mol. Cancer Ther 15(5), 971-84.
6. Vasyutina E., …, Herling M. (2015). The regulatory in-teraction of EVI1 with the TCL1A oncogene impacts cell survival and clinical outcome in CLL. Leukemia 29(10), 2003-14.
7. Prinz C., Vasyutina E., … Herling M. (2015). Organo-metallic nucleosides induce non-classical leukemic cell death that is mitochondrial-ROS dependent and facili-tated by TCL1-oncogene burden. Mol. Cancer 14, 114.
Pflug, N., Cramer, P., Robrecht, S., Bahlo, J., Westermann, A., Fink, A.M., Schrader, A., Mayer, P., Oberbeck, S., Seiler, T., Zenz, T., Durig, J., Kreuzer, K.A., Stilgenbauer, S., Eichhorst, B., Hallek, M., Herling, M., and Hopfinger, G. (2019). New lessons learned in T-PLL: results from a prospective phase-II trial with fludarabine-mitoxantrone-cyclophosphamide-alemtuzumab induction followed by alemtuzumab maintenance. Leuk Lymphoma 60, 649-57.
Schrader, A., Braun, T., and Herling, M. (2019). The dawn of a new era in treating T-PLL. Oncotarget 10, 626-8.
Staber, P.B., Herling, M., Bellido, M., Jacobsen, E.D., Davids, M.S., Kadia, T.M., Shustov, A., Tournilhac, O., Bachy, E., Zaja, F., Porkka, K., Hoermann, G., Simonitsch, I., Haferlach, C., Kubicek, S., Mayerhoefer, M., Hopfinger, G., Jaeger, U., and Dearden, C. (2019). Consensus criteria for diagnosis, staging, and treatment response assessment of T-cell prolymphocytic leukemia (T-PLL). Blood10.1182/blood.2019000402.
Andersson EI, Putzer S, Yadav B, Dufva O, Khan S, He L, Sellner L, Schrader A, Crispatzu G, Oles M, Zhang H, Adnan-Awad S, Lagstrom S, Bellanger D, Mpindi JP, Eldfors S, Pemovska T, Pietarinen P, Lauhio A, Tomska K, Cuesta-Mateos C, Faber E, Koschmieder S, Brummendorf TH, Kytola S, Savolainen ER, Siitonen T, Ellonen P, Kallioniemi O, Wennerberg K, Ding W, Stern MH, Huber W, Anders S, Tang J, Aittokallio T, Zenz T, Herling M, and Mustjoki S (2018). Discovery of novel drug sensitivities in T-PLL by high-throughput ex vivo drug testing and mutation profiling. Leukemia 32, 774-787.
Kotrova M, Novakova M, Oberbeck S, Mayer P, Schrader A, Knecht H, Hrusak O, Herling M, and Bruggemann M (2018). Next-generation amplicon TRB locus sequencing can overcome limitations of flow-cytometric Vbeta expression analysis and confirms clonality in all T-cell prolymphocytic leukemia cases. Cytometry A 93, 1118-1124.
Meder L, Schuldt P, Thelen M, Schmitt A, Dietlein F, Klein S, Borchmann S, Wennhold K, Vlasic I, Oberbeck S, Riedel R, Florin A, Golfmann K, Schlosser HA, Odenthal M, Buttner R, Wolf J, Hallek M, Herling M, von Bergwelt-Baildon M, Reinhardt HC, and Ullrich RT (2018). Combined VEGF and PD-L1 blockade displays synergistic treatment effects in an autochthonous mouse model of small cell lung cancer. Cancer Res10.1158/0008-5472.CAN-17-2176.
Pflug N, Cramer P, Robrecht S, Bahlo J, Westermann A, Fink AM, Schrader A, Mayer P, Oberbeck S, Seiler T, Zenz T, Durig J, Kreuzer KA, Stilgenbauer S, Eichhorst B, Hallek M, Herling M, and Hopfinger G (2018). New lessons learned in T-PLL: results from a prospective phase-II trial with fludarabine-mitoxantrone-cyclophosphamide-alemtuzumab induction followed by alemtuzumab maintenance. Leuk Lymphoma10.1080/10428194.2018.1488253, 1-9.
Schrader A, Crispatzu G, Oberbeck S, Mayer P, Putzer S, von Jan J, Vasyutina E, Warner K, Weit N, Pflug N, Braun T, Andersson EI, Yadav B, Riabinska A, Maurer B, Ventura Ferreira MS, Beier F, Altmuller J, Lanasa M, Herling CD, Haferlach T, Stilgenbauer S, Hopfinger G, Peifer M, Brummendorf TH, Nurnberg P, Elenitoba-Johnson KSJ, Zha S, Hallek M, Moriggl R, Reinhardt HC, Stern MH, Mustjoki S, Newrzela S, Frommolt P, and Herling M (2018). Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL. Nat Commun 9, 697.
Andersson EI, Putzer S, Yadav B, Dufva O, Khan S, He L, Sellner L, Schrader A, Crispatzu G, Oles M, Zhang H, Adnan-Awad S, Lagstrom S, Bellanger D, Mpindi JP, Eldfors S, Pemovska T, Pietarinen P, Lauhio A, Tomska K, Cuesta-Mateos C, Faber E, Koschmieder S, Brummendorf TH, Kytola S, Savolainen ER, Siitonen T, Ellonen P, Kallioniemi O, Wennerberg K, Ding W, Stern MH, Huber W, Anders S, Tang J, Aittokallio T, Zenz T, Herling M, and Mustjoki S (2017). Discovery of novel drug sensitivities in T-PLL by high-throughput ex vivo drug testing and mutation profiling. Leukemia 10.1038/leu.2017.252.
Crispatzu G, Kulkarni P, Toliat MR, Nurnberg P, Herling M, Herling CD, and Frommolt P (2017). Semi-automated cancer genome analysis using high-performance computing. Hum Mutat 38, 1325-35.
Herling M, Rengstl B, Scholtysik R, Hartmann S, Kuppers R, Hansmann ML, Diebner HH, Roeder I, Abken H, Newrzela S, and Kirberg J (2017). Concepts in mature T-cell lymphomas - highlights from an international joint symposium on T-cell immunology and oncology. Leuk Lymphoma 58, 788-96.
Knittel G, Rehkamper T, Korovkina D, Liedgens P, Fritz C, Torgovnick A, Al-Baldawi Y, Al-Maarri M, Cun Y, Fedorchenko O, Riabinska A, Beleggia F, Nguyen PH, Wunderlich FT, Ortmann M, Montesinos-Rongen M, Tausch E, Stilgenbauer S, L PF, Herling M, Herling C, Bahlo J, Hallek M, Peifer M, Buettner R, Persigehl T, and Reinhardt HC (2017). Two mouse models reveal an actionable PARP1 dependence in aggressive chronic lymphocytic leukemia. Nat Commun 8, 153.
Rengstl B, Kim S, Doring C, Weiser C, Bein J, Bankov K, Herling M, Newrzela S, Hansmann ML, and Hartmann S (2017). Small and big Hodgkin-Reed-Sternberg cells of Hodgkin lymphoma cell lines L-428 and L-1236 lack consistent differences in gene expression profiles and are capable to reconstitute each other. PLoS One 12, e0177378.
Shimabukuro-Vornhagen A, Garcia-Marquez M, Fischer RN, Iltgen-Breburda J, Fiedler A, Wennhold K, Rappl G, Abken H, Lehmann C, Herling M, Wolf D, Fatkenheuer G, Rubbert-Roth A, Hallek M, Theurich S, and von Bergwelt-Baildon M (2017). Antigen-presenting human B cells are expanded in inflammatory conditions. J Leukoc Biol 101, 577-87.
Information from this funding period will not be updated anymore. New research related information is available here.
Q. Jiang (PhD student)
J. Stachelscheid (PhD student)
A. Schrader (PostDoc)
P. Mayer (technician)
J. von Jan (PhD student)
T. Braun (Medical student)
L. Wahnschaffe (Medical student)
D. Jungherz (PostDoc)
A. Kondo-Ados (PhD student)
I. Tzianopoulos (PhD student)
E. Weith (PhD student)