Page 104 - 2019_06-Haematologica-web

A. Agraz-Doblas et al.
whether or not MLL-AF4 by itself is sufficient to initiate BCP-ALL in humans. The silent mutational landscape observed in this study and by others20 certainly votes in favor of MLL-AF4+ iBCP-ALL being initiated by a single “big-bang” transformation hit, probably in a short-lived but highly proliferative prenatal B-cell progenitor.4 This hypothesis is supported by recent work by Lin et al., who indeed demonstrated that enforced expression of a fusion transcript consisting of human MLL and murine Af4 in cord blood-derived CD34+ HPSC is sufficient to induce pro-B ALL in xenografted immunodeficient mice.21,22 Yet, similar results using a human MLL-AF4 transcript remain to be established.
Although MLL-AF4 by itself may be sufficient to induce BCP-ALL without significant contributions from coopera- tive genetic lesions, the contribution of the MLL-AF4 and RAS mutations to leukemogenesis should take into account the nature of both the fetal target cell for transfor- mation and the leukemia-initiating cell, according to the increasingly accepted stochastic stem cell model of B- ALL.46,47 Here, we employed high-throughput BCR- sequencing of the IgH locus to delineate the dynamics of clonality of B-cell populations in paired diagnosis-relapse samples of t(4;11)/MLL-AF4+ iBCP-ALL. While pediatric patients with E2A-PBX1+, TEL-AML1+ and BCR-ABL1+ B- ALL all had significantly clonal disease, with a major VDJ rearranged B-cell IgH clone accounting for up to 40% of all BCR, infants with MLL-AF4+ BCP-ALL exhibited a BCR repertoire composed of thousands of minor, non-expanded VDJ rearranged IgH B-cell clones. Because MLL fusions are clonal and RAS mutations are found in clones of relative big size, this suggests that MLL fusions with or without RAS mutations are likely to originate in primitive fetal pro- genitors that have a germline or an incompletely rearranged (DJ) IgH locus.48 Indeed, an unsupervised com- parison of the transcriptome of FL HSPC populations and iBCP-ALL blasts suggests that while the gene expression of primitive FL HSPC (Lin-CD38-CD34+CD19- populations) is similar to that of iBCP-ALL, FL B progenitors (CD34+CD19+) are transcriptionally distinct. Our data ele- gantly reinforces previous fluorescence in-situ hybridiza- tion findings suggesting that a primitive “pre-VDJ” stem/progenitor cell (perhaps CD34+CD19-) may represent the cell in which both t(4;11) and RAS mutations arise.14,31,49
Cooperative leukemogenic events in iBCP-ALL may need to be sought beyond genetic insults; for instance, epi- genetic and transcriptomic deregulation. MLL-AF4 might only induce BCP-ALL in cells that meet certain epigenetic and transcriptomic make-up criteria, either influenced by microenvironmental cues, or characteristic of the cell-of- origin.31 Indeed, lesions such as RAS mutations may con- tribute to disease pathogenesis only against certain intrin- sic epigenetic or transcriptomic backgrounds present in the cell in which the MLL translocations occurred50,51. This is supported by the limited impact of RAS mutations in transcriptomic signatures associated with leukemia origin, development and pathogenesis, although this is likely due to the subclonal nature of RAS mutations.38 However, in line with the reported contribution of RAS mutations to extramedullary infiltration of MLLr BCP-ALL blasts,27 RAS-mutated patients displayed a transcriptomic signa- ture associated with migration.
The functional and molecular contribution of the recip- rocal fusion genes resulting from the derivative translo- cated chromosomes remains obscure in cancer. The AF4-
MLL genomic fusion was previously detected in 80-85% of t(4;11)+ patients.5,52 Our “multi-layered omics” approach allowed for the exact characterization of the t(4;11) molecular DNA/RNA break points and the identi- fication of those patients expressing the reciprocal AF4- MLL fusion. We now report that the AF4-MLL reciprocal fusion is expressed in only 50% of t(4;11)+ iBCP-ALL patients. Strikingly, there was a previously unrecognized and very significant positive correlation between the upregulation of the HOXA gene cluster and the expres- sion of AF4-MLL. Of note, a recent study showed that approximately half of t(4;11)+ patients do not have an activated HOXA signature.44,53,54 Furthermore, in the recent MLL-Af4-induced B-ALL xenograft model MLL- Af4 failed to bind to HOXA genes and therefore HOXA gene expression was not upregulated.21 This is experi- mentally supported by chromatin immunoprecipitation- sequencing analysis performed in human embryonic stem cells transduced with MLL-AF4, AF4-MLL or both show- ing a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in dou- ble fusion-expressing hematopoietic derivatives, estab- lishing a functional and molecular cooperation between MLL-AF4 and AF4-MLL fusions during human hematopoietic development (data not shown). Strikingly, AF4-MLL-expressing patients had a 5-fold longer event- free survival and a 3-fold longer overall survival compared to t(4;11)+ iBCP-ALL patients lacking AF4-MLL expres- sion, which is in line with previous reports suggesting that high HOXA gene expression is associated with improved survival and lower risk of relapse.22,39 Because the expression of AF4-MLL is not analyzed in routine molecular diagnosis, our “multi-layered omics” approach was critical to unraveling the association between AF4- MLL and HOXA expression, thus identifying a novel sub- group of t(4;11)+ iBCP-ALL with better clinical outcome. It is very important for routine diagnostic and clinical practice that when the expression of AF4-MLL was eval- uated in a Cox model adjusting for risk stratification (medium risk or high risk according to the Interfant-06 protocol), it retained its prognostic significance.
Mechanistically, AF4-MLL contains the SET domain dis- rupted from its "specification domain", the N-terminal portion of MLL, which binds to MEN1 and LEDGF thus shaping the gene-targeting module of the MLL gene. When AF4-MLL is expressed, the N-terminal portion is substituted by the AF4 N-terminus (AF4N) which is the crucial domain for binding to and strongly activating RNA polymerase II (RNAP II) for transcriptional elongation. Thus, expression of A4M-MLL may induce robust RNAP II-dependent gene transcription by overwriting the elon- gation control process in a dominant fashion.55–58 We hypothesize that a likely function of AF4-MLL could be to prepare the ground for MLL-AF4 or other transcription factors to skew normal and leukemic hematopoietic cell fate decisions. This also explains why MLL-AF4, but not AF4-MLL, seems to be necessary in 100% of patients.
Despite being a developmental cancer, iBCP-ALL patients did not show reactivation of pluripotent or embryonic-like gene expression signatures as revealed by RNA-sequencing. Additional research is required to deci- pher the nature of the insults initiating MLLr iBCP-ALL, as so far we can only speculate on the data currently avail- able. Whole-genome pyrosequencing will likely provide unique insights into the DNA methylome landscape of
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haematologica | 2019; 104(6)