Inactivation of Stage-Specific B-Cell Commitment Genes Generates Distinct Molecular Subtypes of BCR-ABL1 Lymphoblastic Leukemia

Background Philadelphia-chromosome positive acute lymphoblastic leukemia (Ph+ ALL), the most common form of ALL in adults, is a highly aggressive blood malignancy defined by the BCR-ABL1 fusion. Although inhibitors targeting the BCR-ABL1 oncoprotein, such as imatinib, have significantly improved clinical response rates for this disease, a subset of patients are refractory to therapy or respond initially but relapse soon after. ABL1 kinase domain mutations partly explain differential responses in patients; however, for the majority of cases, a molecular basis that can reconcile this clinical observation is lacking.

Methods Flow-sorted blasts from 53 primary samples, 49 de novo Ph+ ALL and 4 lymphoid blast crisis CML, were subjected to RNA sequencing (RNA-seq) and whole genome sequencing (WGS). Response rates were tracked using BCR-ABL1 transcript levels from patient blood.

Results Hierarchical clustering of transcriptome data produced two molecular subgroups of Ph+ ALL. One subgroup, which we termed 'Core-B', upregulated key regulators of B-lymphoid differentiation including IL7R and MS4A1 (CD20). By contrast, the second subgroup upregulated an expression program related to hematopoietic stem cell (HSC) and myeloid differentiation, with upregulation of KIT, CD34, MPO, CSF3R, and GATA3. We termed this subgroup 'Aberrant-Stem-Myeloid' (ASM). These subgroups displayed a striking disparity in response rates to intensive chemotherapy with imatinib. Whereas 'Core-B' patients showed highly durable responses often lasting many years, 'ASM' patients frequently relapsed (4% vs 43% relapse; p=0.007). We used WGS analysis to investigate the genetic basis of these molecular subtypes. 'Core-B' Ph+ ALLs were enriched for deletions in PAX5, a B-cell specification gene, and CDKN2A/B, tumor suppressors. The 'ASM' subtype lacked these genetic alterations; instead, these leukemias were enriched for deletions in EBF1, an early B-cell lineage factor that represses T-lymphoid and myeloid lineages and is expressed before PAX5 in B-cell lineage differentiation. Accordingly, blasts from 'ASM' leukemias with EBF1 deletions showed decreased CD19 antigen expression and upregulation of myeloid antigens by clinical flow cytometry. Rare cases with concurrent EBF1 and PAX5 deletions showed expression features of both 'ASM' and 'Core-B' leukemias. Mutations observed in myeloid leukemias (TET2, RUNX1) were only present in the 'ASM' subtype. Loss of IKZF1, found in 77% of cases, also displayed distinct patterns between the two subgroups; deletions leading to the dominant negative isoform (Ik6) were enriched in the 'Core-B' subgroup (45% vs. 14%; p=0.019) while monosomy 7 and large deletions encompassing IKZF1 were enriched in the 'ASM' leukemias (41% vs. 10%; p=0.017). In 1 of 4 diagnosis/relapse patients analyzed, a molecular switch from 'Core-B' at diagnosis to 'ASM' at relapse was observed. The diagnostic 'Core-B' clone from this patient harbored a PAX5 mutation that was lost at relapse, whereas the relapsed 'ASM' clone harbored trisomy 21 and a RUNX1 mutation. Altogether, our data suggest that the 'ASM' leukemias emerge through dysregulation of genes earlier in lympho-myeloid specification compared to 'Core-B' leukemias.

These findings led us to investigate if the 'ASM' subtype originates from an HSC and the 'Core-B' subtype originates from a B-cell progenitor. We first looked at the distribution of the long (p210) and short (p190) isoforms of BCR-ABL1 in the two subtypes. The p210 isoform, also the hallmark of CML, is speculated to arise in an HSC, and the p190 is thought to arise in a B-cell progenitor. Neither the p190 or p210 BCR-ABL1 isoform was enriched in either subgroup. We resolved highly purified HSC and progenitor subsets from CD34+CD19- cells, functionally evaluated by methylcellulose assays, and subjected them to a sensitive nested-PCR strategy. Cases from both the 'ASM' and 'Core-B' subtypes showed HSC/myeloid progenitor involvement regardless of the BCR-ABL1 isoform. This data suggest that the cell-of-origin does not play a role in establishing the molecular subtype of leukemia blasts.

Conclusion There are two distinct molecular subtypes of Ph+ ALL that demonstrate differential responses to treatment and emerge from independent mutational routes. Moreover, the key genetic determinants that form the molecular subtype are secondary driver alterations that lie downstream of BCR-ABL1.