The variable clinical course in chronic lymphocytic leukaemia (CLL) largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We show that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling regulator and its high levels are associated with worse CLL prognosis. We identified microRNA (miRNA) miR-34a as the most prominently upregulated miRNA during DDR in CLL cells in vitro and in vivo during FCR therapy (fludarabine, cyclophosphamide, rituximab). MiR-34a induced by DDR activation and p53 stabilization potently represses FOXP1 expression by binding in its 3'-UTR. The low FOXP1 levels limit BCR signalling partially via derepressing BCR-inhibitory molecule CD22. We also show that low miR-34a levels can be used as a biomarker for worse response or shorter progression free survival in CLL patients treated with FCR chemoimmunotherapy, and shorter overall survival, irrespective of TP53 status. Additionally, we have developed a method for the absolute quantification of miR-34a copies and defined precise prognostic/predictive cutoffs. Overall, herein, we reveal for the first time that B cells limit their BCR signalling during DDR by down-modulating FOXP1 via DDR-p53/miR-34a axis.

Bioinformatics and Genomics Unit MBC Centro di Biotecnologie Molecolari Torino Italy

Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine MU Brno Czech Republic

Genomics Core Facility EMBL Heidelberg Heidelberg Germany

Molecular Medicine CEITEC MU Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science MU Brno Czech Republic

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Byrd JC, Furman RR, Coutre SE, Flinn IW, Burger JA, Blum KA, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med. 2013;369:32–42. DOI

Furman RR, Cheng S, Lu P, Setty M, Perez AR, Guo A, et al. Ibrutinib resistance in chronic lymphocytic leukemia. N Engl J Med. 2014;370:2352–4. DOI

Hallek M, Fischer K, Fingerle-Rowson G, Fink A, Busch R, Mayer J, et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. 2010;376:1164–74. DOI

Döhner H, Stilgenbauer S, Benner A, Leupolt E, Kröber A, Bullinger L, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343:1910–6. DOI

Trbusek M, Smardova J, Malcikova J, Sebejova L, Dobes P, Svitakova M, et al. Missense mutations located in structural p53 DNA-binding motifs are associated with extremely poor survival in chronic lymphocytic leukemia. J Clin Oncol. 2011;29:2703–8. DOI

Malcikova J, Pavlova S, Kozubik KS, Pospisilova S. TP53 mutation analysis in clinical practice: lessons from chronic lymphocytic leukemia. Hum Mutat. 2014;35:663–71. DOI

D’Avola A, Drennan S, Tracy I, Henderson I, Chiecchio L, Larrayoz M, et al. Surface IgM expression and function associate with clinical behavior, genetic abnormalities and DNA methylation in CLL. Blood. 2016;128:816–26. DOI

Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham G. B-cell receptor signaling in chronic lymphocytic leukemia. Blood. 2011;118:4313–20. DOI

Kipps TJ. The B-cell receptor and ZAP-70 in chronic lymphocytic leukemia. Best Pract Res Clin Haematol. 2007;20:415–24. DOI

Seda V, Mraz M. B-cell receptor signalling and its crosstalk with other pathways in normal and malignant cells. Eur J Haematol. 2015;94:193–205. DOI

Cui B, Chen L, Zhang S, Mraz M, Fecteau J-F, Yu J, et al. MicroRNA-155 influences B-cell receptor signaling and associates with aggressive disease in chronic lymphocytic leukemia. Blood. 2014;124:546–54. DOI

Nakade K, Zheng H, Ganguli G, Buchwalter G, Gross C, Wasylyk B. The tumor suppressor p53 inhibits Net, an effector of Ras/extracellular signal-regulated kinase signaling. Mol Cell Biol. 2004;24:1132–42. DOI

Stambolic V, MacPherson D, Sas D, Lin Y, Snow B, Jang Y, et al. Regulation of PTEN transcription by p53. Mol Cell. 2001;8:317–25. DOI

Elston R, Inman GJ. Crosstalk between p53 and TGF- β Signalling. J Signal Transduct. 2012;2012:1–10. DOI

Mraz M, Chen L, Rassenti LZ, Ghia EM, Li H, Jepsen K, et al. miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1. Blood. 2014;124:84. DOI

Barrans SL, Fenton JAL, Banham A, Owen RG, Jack AS. Strong expression of FOXP1 identifies a distinct subset of diffuse large B-cell lymphoma (DLBCL) patients with poor outcome. Blood. 2004;104:2933–5. DOI

Sagaert X, de Paepe P, Libbrecht L, Vanhentenrijk V, Verhoef G, Thomas J, et al. Forkhead box protein P1 expression in mucosa-associated lymphoid tissue lymphomas predicts poor prognosis and transformation to diffuse large B-cell lymphoma. J Clin Oncol. 2006;24:2490–7. DOI

Brown P, Marafioti T, Kusec R, Banham AH. The FOXP1 transcription factor is expressed in the majority of follicular lymphomas but is rarely expressed in classical and lymphocyte predominant hodgkin’s lymphoma. J Mol Histol. 2005;36:249–56. DOI

van Keimpema M, Grüneberg LJ, Mokry M, van Boxtel R, Koster J, Coffer PJ, et al. FOXP1 directly represses transcription of proapoptotic genes and cooperates with NF-κB to promote survival of human B cells. Blood. 2014;124:3431–40. DOI

Dekker JD, Park D, Shaffer AL, Kohlhammer H, Deng W, Lee B-K, et al. Subtype-specific addiction of the activated B-cell subset of diffuse large B-cell lymphoma to FOXP1. Proc Natl Acad Sci USA. 2016;113:E577–E586. DOI

van Keimpema M, Grüneberg LJ, Schilder-Tol EJM, Oud MECM, Beuling EA, Hensbergen PJ, et al. The small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma and full-length FOXP1 exert similar oncogenic and transcriptional activity in human B cells. Haematologica. 2017;102:573–83. DOI

Walker MP, Stopford CM, Cederlund M, Fang F, Jahn C, Rabinowitz AD, et al. FOXP1 potentiates Wnt/β-catenin signaling in diffuse large B cell lymphoma. Sci Signal. 2015;8:ra12. nor DOI

Sagardoy A, Martinez-Ferrandis JI, Roa S, Bunting KL, Aznar MA, Elemento O, et al. Downregulation of FOXP1 is required during germinal center B-cell function. Blood. 2013;121:4311–20. DOI

van Boxtel R, Gomez-Puerto C, Mokry M, Eijkelenboom A, van der Vos KE, Nieuwenhuis EE, et al. FOXP1 acts through a negative feedback loop to suppress FOXO-induced apoptosis. Cell Death Differ. 2013;20:1219–29. DOI

Flori M, Schmid CA, Sumrall ET, Tzankov A, Law CW, Robinson MD, et al. The hematopoietic oncoprotein FOXP1 promotes tumor cell survival in diffuse large B-cell lymphoma by repressing S1PR2 signaling. Blood. 2016;127:1438–48. DOI

Musilova K, Mraz M. MicroRNAs in B-cell lymphomas: how a complex biology gets more complex. Leukemia. 2015;29:1004–17. DOI

Mraz M, Kipps TJ. MicroRNAs and B cell receptor signaling in chronic lymphocytic leukemia. Leuk Lymphoma. 2013;54:1836–9. DOI

Vigorito E, Perks KL, Abreu-Goodger C, Bunting S, Xiang Z, Kohlhaas S, et al. microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells. Immunity. 2007;27:847–59. DOI

Mraz M, Dolezalova D, Plevova K, Stano Kozubik K, Mayerova V, Cerna K, et al. MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia. Blood. 2012;119:2110–3. DOI

Mraz M, Malinova K, Kotaskova J, Pavlova S, Tichy B, Malcikova J, et al. miR-34a, miR-29c and miR-17-5p are downregulated in CLL patients with TP53 abnormalities. Leukemia. 2009;23:1159–63. DOI

Zenz T, Mohr J, Eldering E, Kater AP, Bühler A, Kienle D, et al. miR-34a as part of the resistance network in chronic lymphocytic leukemia. Blood. 2009;113:3801–8. DOI

Asslaber D, Pinon JD, Seyfried I, Desch P, Stocher M, Tinhofer I, et al. microRNA-34a expression correlates with MDM2 SNP309 polymorphism and treatment-free survival in chronic lymphocytic leukemia. Blood. 2010;115:4191–7. DOI

Dufour A, Palermo G, Zellmeier E, Mellert G, Duchateau-Nguyen G, Schneider S, et al. Inactivation of TP53 correlates with disease progression and low miR-34a expression in previously treated chronic lymphocytic leukemia patients. Blood. 2013;121:3650–7. DOI

Chen L, Widhopf G, Huynh L, Rassenti L, Rai KR, Weiss A, et al. Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia. Blood. 2002;100:4609–14. DOI

Mottok A, Jurinovic V, Farinha P, Rosenwald A, Leich E, Ott G, et al. FOXP1 expression is a prognostic biomarker in follicular lymphoma treated with rituximab and chemotherapy. Blood. 2018;131:226–35. DOI

Banham AH, Connors JM, Brown PJ, Cordell JL, Ott G, Sreenivasan G, et al. Expression of the FOXP1 transcription factor is strongly associated with inferior survival in patients with diffuse large B-cell lymphoma. Clin Cancer Res J Am Assoc Cancer Res. 2005;11:1065–72.

Brown PJ, Ashe SL, Leich E, Burek C, Barrans S, Fenton JA, et al. Potentially oncogenic B-cell activation–induced smaller isoforms of FOXP1 are highly expressed in the activated B cell–like subtype of DLBCL. Blood. 2008;111:2816–24. DOI

Cejkova S, Rocnova L, Potesil D, Smardova J, Novakova V, Chumchalova J, et al. Presence of heterozygous ATM deletion may not be critical in the primary response of chronic lymphocytic leukemia cells to fludarabine. Eur J Haematol. 2009;82:133–42. DOI

He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y, et al. A microRNA component of the p53 tumour suppressor network. Nature. 2007;447:1130–4. DOI

Christoffersen NR, Shalgi R, Frankel LB, Leucci E, Lees M, Klausen M, et al. p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC. Cell Death Differ. 2010;17:236. DOI

Vinall RL, Ripoll AZ, Wang S, Pan C-X, deVere White RW. MiR-34a chemosensitizes bladder cancer cells to cisplatin treatment regardless of p53-Rb pathway status. Int J Cancer. 2012;130:2526–38. DOI

Macleod KF, Sherry N, Hannon G, Beach D, Tokino T, Kinzler K, et al. p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev. 1995;9:935–44. DOI

Galanos P, Vougas K, Walter D, Polyzos A, Maya-Mendoza A, Haagensen EJ, et al. Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing. Nat Cell Biol. 2016;18:777–89. DOI

Nitschke L, Carsetti R, Ocker B, Köhler G, Lamers MC. CD22 is a negative regulator of B-cell receptor signalling. Curr Biol. 1997;7:133–43. DOI

Zenz T, Habe S, Denzel T, Mohr J, Winkler D, Buhler A, et al. Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood. 2009;114:2589–97. DOI

Bartels CL, Tsongalis GJ. MicroRNAs: novel biomarkers for human cancer. Clin Chem. 2009;55:623–31. DOI

Antonini D, Russo MT, De Rosa L, Gorrese M, Del Vecchio L, Missero C. Transcriptional repression of miR-34 family contributes to p63-mediated cell cycle progression in epidermal cells. J Invest Dermatol. 2010;130:1249–57. DOI

Kato S, Han S-Y, Liu W, Otsuka K, Shibata H, Kanamaru R, et al. Understanding the function–structure and function–mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis. Proc Natl Acad Sci USA. 2003;100:8424–9. DOI

Pulikkan JA, Peramangalam PS, Dengler V, Ho PA, Preudhomme C, Meshinchi S, et al. C/EBP regulated microRNA-34a targets E2F3 during granulopoiesis and is down-regulated in AML with CEBPA mutations. Blood. 2010;116:5638–49. DOI

Wang LQ, Kwong YL, Wong KF, Kho CSB, Jin DY, Tse E, et al. Epigenetic inactivation of mir-34b/c in addition to mir-34a and DAPK1 in chronic lymphocytic leukemia. J Transl Med. 2014;12:52. DOI

Craig VJ, Cogliatti SB, Imig J, Renner C, Neuenschwander S, Rehrauer H, et al. Myc-mediated repression of microRNA-34a promotes high-grade transformation of B-cell lymphoma by dysregulation of FoxP1. Blood. 2011;117:6227–36. DOI

Jens M, Rajewsky N. Competition between target sites of regulators shapes post-transcriptional gene regulation. Nat Rev Genet. 2015;16:113–26. DOI

Deneberg S, Kanduri M, Ali D, Bengtzen S, Karimi M, Qu Y, et al. microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia. Epigenetics. 2014;9:910–7. DOI

Bader AG. miR-34 – a microRNA replacement therapy is headed to the clinic. Front Genet Front Genet. 2012;3:120. PubMed

Le Garff-Tavernier M, Blons H, Nguyen-Khac F, Pannetier M, Brissard M, Gueguen S, et al. Functional assessment of p53 in chronic lymphocytic leukemia. Blood Cancer J. 2011;1:e5–e5. DOI

Pozzo F, Dal BoM, Peragine N, Bomben R, Zucchetto A, Rossi FM, et al. Detection of TP53 dysfunction in chronic lymphocytic leukemia by an in vitro functional assay based on TP53 activation by the non-genotoxic drug Nutlin-3: a proposal for clinical application. J Hematol Oncol J Hematol Oncol. 2013;6:83. DOI

te Raa GD, Malcikova J, Mraz M, Trbusek M, Garff-Tavernier L, Merle-Beral H, et al. Assessment of TP53 functionality in chronic lymphocytic leukaemia by different assays; an ERIC-wide approach. Br J Haematol. 2014;167:565–9. DOI

te Raa GD, Moerland PD, Leeksma AC, Derks IA, Yigittop H, Laddach N, et al. Assessment of p53 and ATM functionality in chronic lymphocytic leukemia by multiplex ligation-dependent probe amplification. Cell Death Dis. 2015;6:e1852–e1852. DOI

Urgard E, Brjalin A, Langel Ü, Pooga M, Rebane A, Annilo T. Comparison of peptide- and lipid-based delivery of miR-34a-5p Mimic into PPC-1 Cells. Nucleic Acid Ther. 2017;27:295–302. DOI

Trang P, Wiggins JF, Daige CL, Cho C, Omotola M, Brown D, et al. Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol Ther. 2011;19:1116–22. DOI

MicroRNA Profiling of Self-Renewing Human Neural Stem Cells Reveals Novel Sets of Differentially Expressed microRNAs During Neural Differentiation In Vitro

FoxO1-GAB1 axis regulates homing capacity and tonic AKT activity in chronic lymphocytic leukemia

miR-29 modulates CD40 signaling in chronic lymphocytic leukemia by targeting TRAF4: an axis affected by BCR inhibitors

LncRNAs in adaptive immunity: role in physiological and pathological conditions

Genetic and Non-Genetic Mechanisms of Resistance to BCR Signaling Inhibitors in B Cell Malignancies

Oct4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts