Evidence for a role of RUNX1 as recombinase cofactor for TCRβ rearrangements and pathological deletions in ETV6-RUNX1 ALL

Abstract

T-cell receptor gene beta (TCRβ) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knockout mouse model and demonstrate by deep TCRβ sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRβ rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRβ gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRβ gene rearrangements in TALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1 +/− mouse model, heterozygous Runx1 mutations in TALL patients displayed no detectable impact on TCRβ rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions. RUNX1 belongs to the evolutionary conserved Runt transcription factor family and is indispensable for the establishment of definitive hematopoiesis in vertebrates and is an important regulator of cells of the immune system 1-3. RUNX1 is among the most frequently mutated genes in various hematological malignancies and RUNX1 alterations can lead to a loss of RUNX1 function or to a dominant-negative effect 4,5. Mono-allelic RUNX1 mutations occur in approximately 15% of T-Cell Acute Lymphoblastic Leukemia (T-ALL), predominantly in cases with an immature phenotype and a poor prognosis 6-8. In de novo Acute Myeloid Leukemia (AML) patients, somatic mutations in RUNX1 are detectable in approximately 3% of children and 15% of adults 4. In an AML subgroup with an immature phenotype (AML-M0), 30% of the cases are associated with bi-allelic inactivating RUNX1 point mutations and deletions 9. Patients with Myelodysplastic Syndrome (MDS) carrying RUNX1 mutations have a higher risk and shorter latency for progression to AML 10. Furthermore, there are over 50 different types of chromosomal translocations affecting RUNX1 4. We focus in this study on the most common translocation, t(12;21), occurring in approximately 20% of childhood B-cell

Treatment with ribociclib shows favourable immunomodulatory effects in patients with hormone receptor-positive breast cancer—findings from the RIBECCA trial

“…To analyse changes in the TCR repertoire, deep TCR profiling was performed at the time points W0 and W12 of ribociclib treatment using next-generation sequencing. For this, cDNA was synthesised from isolated total RNA of patients' PBMCs with the GoScript™ Reverse Transcriptase System (Promega, Madison, WI, USA), after which a TCRsafe™ analysis (HS Diagnomics, Berlin, Germany) was carried out to quantitatively analyse the TCRβ repertoire by next-generation sequencing as described previously [19].…”