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A. Agraz-Doblas et al.
3D). This indicates that infants with MLL-AF4+ BCP-ALL relapse irrespective of the status of KRAS and NRAS. Thus, subclones carrying KRAS mutations do not exert an advantage over non-mutated clones, despite representing a recurrent genetic insult at diagnosis. Hence, this would argue against a leukemia-initiating role for RAS mutations.38 Alternatively, RAS mutations might indeed be leukemogenic drivers, but the treatment-induced genetic instability observed at relapse may compensate de novo RAS mutations, acting as new leukemia drivers coop- erating with MLL-AF4 during relapse.
HOXA cluster genes are only expressed in t(4;11)+ patients expressing the reciprocal fusion AF4-MLL which determines clinical outcome
To gain insights into the mechanisms underlying leuke- mogenesis in these mutationally silent MLLr and MLL germline iBCP-ALL patients, we performed RNA- sequencing in the discovery cohort of patients (n=42) using FL-derived CD34+CD19+ healthy B-cell progenitors as controls, as these cells most likely represent the healthy counterparts of the leukemic blast stalled at the pro/pre-B- cell differentiation stage. We first surveyed the expression of the genes previously reported to be specific to either MLLr iBCP-ALL or specifically to MLL-AF4+ iBCP-ALL39. RNA-sequencing profiling confirmed that these genes seg- regate patients according to the molecular subtype, MLL- AF4+, MLL-AF9+ and MLL germline (Online Supplementary Figure S7). We also observed, at diagnosis, a strong upreg- ulation of the MLL target genes FLT3,40 MEIS1, PROM1 and HOXA genes in many of our MLLr iBCP-ALL samples but not in MLL germline samples (t-test, P<0.05) (Figure 4A), thus validating our RNA-sequencing approach.
Strikingly, the reciprocal AF4-MLL fusion gene was dis- cernibly expressed in 19/43 (45%) of the t(4;11)+ iBCP- ALL samples, and its expression was always maintained at relapse (data not shown). We then compared the genes dif- ferentially expressed between AF4-MLL-expressing and non-expressing t(4;11)+ patients and found a striking posi- tive correlation between the expression of the HOXA gene cluster and overexpression of the reciprocal AF4-MLL fusion (t-test, P=0.002) (Figure 4B). These AF4- MLL/HOXA-expressing patients (n=19) had a significantly better prognosis than those lacking AF4-MLL/HOXA expression (n=24). Four-year event-free and overall sur- vival rates were 62.4% (SE, 11.3%) versus 11.7% (SE, 10.2%) (P=0.001) (Figure 4C), and 73.7% (SE, 10.1%) ver- sus 25.2% (SE, 10.3%) (P=0.016) (Figure 4D), respectively. When “AF4-MLL expression” was analyzed in a Cox model adjusting for risk stratification (medium risk or high risk according to the Interfant-06 protocol based on age at diagnosis, white blood cell count and response to pred- nisone), it retained its prognostic significance with a haz- ard ratio for patients lacking AF4-MLL expression of 3.42 [95% confidence interval (95% CI): 1.35-8.63; P=0.01) compared to those expressing AF4-MLL/HOXA, while risk group was not significant (HR for high risk vs. medi- um risk, 1.34; 95% CI: 0.59-3.03; P=0.49). This is the first study showing that AF4-MLL overexpression correlates very well with transcriptional deregulation of the HOXA gene cluster in iBCP-ALL and that the co-expression of AF4-MLL and HOXA gene cluster identifies a subgroup of t(4;11)+ iBCP-ALL with a very more favorable clinical out- come.
We next explored new molecular pathways involved in
the pathogenesis of iBCP-ALL, by performing an unbiased transcriptional analysis of the RNA-sequencing data from the iBCP-ALL patients. We found deregulated expression of a total of 3,905 genes, of which 2,575 (66%) were upregulated and 1,330 (34%) downregulated as compared with those of healthy FL-derived B-cell progenitors, illus- trating the global transcriptional activation nature of MLL fusions (Online Supplementary Figure S8).25,41 Furthermore, a significant upregulation of genes involved in the control of cell growth, including the CDK inhibitors P21, P16, P19, P27 and components of the transforming growth factor-b pathway such as TGFB1, SMAD and ACVR1B, was observed in iBCP-ALL (Figure 4D and Online Supplementary Figure S9). By contrast, iBCP-ALL showed a robust downregulation of genes involved in DNA integri- ty checkpoints such as CHEK1, CHEK2, ATM, ATR and RAD17, and in double-strand break repair genes including ERCC4, BRCA1, POLA1 and RAD51 (Figure 4E and Online Supplementary Figure S9). These transcriptional changes were validated by quantitative reverse transcriptase poly- merase chain reaction in ten patients per group (Online Supplementary Figure S10). Deregulation of DNA integrity checkpoints and double-strand break repair genes may well contribute to the genomic instability observed at relapse, and might explain the enrichment in C>T/G>A transitions, associated with the spontaneous deamination of 5-methylcytosine (Online Supplementary Figures S3 and S6).
By using FL-derived normal B-cell progenitors as con- trols, differences between leukemic blasts and their nor- mal counterparts could be identified but this does not allow the definition of transcriptomic differences within the iBCP-ALL cytogenetic groups. We, therefore, analyzed the RNA-sequencing data comparing the genes differen- tially expressed in MLL-AF4+ versus MLL-AF9+ and MLL- wildtype iBCP-ALL patients, without considering normal B-cell progenitors as controls. A gene ontology analysis (gene set enrichment analysis, GSEA) performed with the genes differentially expressed revealed that MLL-AF4+ patients show, as compared to both MLL-AF9+ and MLL- wildtype patients, a significant upregulation of genes asso- ciated with cellular catabolism, coupled to a significant downregulation of negative regulators of the PI3-MAPK pathway, as well as of genes involved in lymphoid differ- entiation and RNApol II transcriptional regulation (Figure 5). This suggests, respectively, a metabolic change in MLL- AF4+ cells towards rapid energy generation while reinforc- ing the basal hyperactivation of the PI3-MAPK pathway by RAS mutations (Figures 1 and 2), a poorly differentiated cellular origin of t(4;11), and an impairment of the normal function of AF4, a key component of the RNApol II tran- scription complex.
Deep-sequencing analysis of B-cell receptor repertoires suggests a hematopoietic stem cell/early pre-VDJ progenitor as the cell-of-origin for t(4;11) and RAS mutations
We next analyzed BCR repertoires to gain insights into the immunoglobulin heavy chain (IgH) rearrangement clonal composition of paired diagnostic-relapse samples from t(4;11)+ iBCP-ALL (4 pairs). BCR are generated through DNA recombination during B-cell differentiation and represent unique markers for each B-cell clone. Because the BCR sequence provides a molecular tag for each B-cell clone, high-throughput sequencing of BCR
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haematologica | 2019; 104(6)