PAX5-driven subtypes of B-progenitor acute lymphoblastic leukemia

Abstract

Recent genomic studies have identified chromosomal rearrangements defining new subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL), however many cases lack a known initiating genetic alteration. Using integrated genomic analysis of 1,988 childhood and adult cases, we describe a revised taxonomy of B-ALL incorporating 23 subtypes defined by chromosomal rearrangements, sequence mutations or heterogeneous genomic alterations, many of which show marked variation in prevalence according to age. Two subtypes have frequent alterations of the B lymphoid transcription-factor gene PAX5. One, PAX5alt (7.4%), has diverse PAX5 alterations (rearrangements, intragenic amplifications or mutations); a second subtype is defined by PAX5 p.Pro80Arg and biallelic PAX5 alterations. We show that p.Pro80Arg impairs B lymphoid development and promotes the development of B-ALL with biallelic Pax5 alteration in vivo. These results demonstrate the utility of transcriptome sequencing to classify B-ALL and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis.

Figure 1.. Integrative B-ALL subtypes.

a, Gene expression profiling (GEP) of 1,988 cases shown in a two-dimensional t-distributed stochastic neighbor embedding (tSNE) plot. Each dot represents a sample. The top 1,000 most variable genes (based on median absolute deviation) were selected and processed by the tSNE algorithm with perplexity score of 30. Major B-ALL subtypes are highlighted in different colors, which include ETV6-RUNX1, KMT2A- (MLL-) rearranged (KMT2A), TCF3-PBX1, DUX4-rearranged (DUX4), ZNF384-rearranged (ZNF384), MEF2D-rearranged (MEF2D), BCR-ABL1 (Ph), Ph-like, High hyperdiploid, Low hypodiploid, Near haploid and cases with intrachromosomal amplification of chromosome 21 (iAMP21). Three uncommon subtypes are also shown: BCL2/MYC-rearranged (BCL2/MYC), TCF3/TCF4-HLF (HLF) and NUTM1-rearranged (NUTM1). A group of samples with distinct GEP and universal PAX5 p.Pro80Arg (P80R) mutation were observed (PAX5 P80R). A cluster of cases with diverse PAX5 alterations (PAX5alt) is also observed adjacent to the PAX5 P80R group, with diverse rearrangements, focal/intragenic amplifications and non-PAX5 P80R mutations. Eight cases with distinct GEP were identified with the same IKZF1 missense mutation p.Asn159Tyr (N159Y). Cases in five subtypes including low hyperdiploid, ETV6-RUNX1-like, KMT2A-like, ZNF384-like and CRLF2(non-Ph-like) are shown as gray dots, but not specifically labeled in the plot. b, The distribution of B-ALL subtypes within each subtype (upper) or each age group (lower). The definition for age groups is described in Table 1. The subtypes are grouped as gross chromosomal alteration, transcription factor (TF) rearrangement, other TF alteration, kinase driven and others.

Figure 2.. Mutational profile of PAX5-altered (PAX5alt) B-ALL

a, Genetic alterations including gene rearrangements (PAX5r), sequence mutations (PAX5mut) and focal intragenic amplifications (PAX5amp) observed in PAX5alt cohort. PAX5 mutation zygosity is defined as: heterozygous (Hetero), MAF <0.8; homozygous (Homo), MAF ≥0.8. For cases with multiple PAX5 mutations, the highest MAF was used to define zygosity. PAX5 copy number alterations (CNA) were called from cases with SNP array data. All the recurrent PAX5 fusions and sequence mutations are shown in the heatmap and the number of cases are indicated in parentheses. The recurrent mutations mean the same reference amino acids are affected, even with different variant amino acids, like p.Arg38Cys and p.Arg38His, are shown as p.Arg38; if the variant amino acids are the same, then the full amino acid changes are shown, e.g. p.Ser66Asn. p.*392Arg is a stop loss mutation. fs, frameshift; sp, canonical splice site mutation. b, Genetic mutation spectrum of 65 PAX5alt cases with whole genome/exome sequencing data. Samples are ordered primarily based on the key PAX5 alterations (PAX5r, PAX5mut and PAX5amp) and genes are grouped into specific pathways. AYA, adolescent and young adult; TF, transcription factor; CN-LOH, copy-neutral loss of heterozygosity; WT, wild type; NA, not available; NME, NOTCH1-driven MYC enhancer.

Figure 3.. Mutational profile of PAX5 P80R B-ALL

a, Protein domain plot of PAX5 showing the 57 mutations detected in 44 patients in PAX5 p.Pro80Arg (P80R) subtype (bottom panel) compared to all the other B-ALL cases (146 mutations in 125 out of 1,944 patients (top panel); which is further divided for PAX5alt and other B-ALL subtypes). Details of the mutations are provided in Supplementary Table 7. Individual cases are represented by circles; missense mutations affecting the same amino acid residues are shown as graded shades of blue to indicate the number of cases for each substitution. b, Copy number alterations (CNAs) identified on chromosome 9 from single nucleotide polymorphism (SNP) array. Two primary target genes CDKN2A and PAX5 affected by CNAs are highlighted. hemi, hemizygous; homo, homozygous; CN-LOH, copy-neutral loss of heterozygosity. c, Genetic mutations including SNVs/Indels and CNAs detected from either transcriptome sequencing, whole genome/exome sequencing or SNP array data in the PAX5 P80R group. Genes are ordered according their recurrence and grouped into specific pathways. Zygosity of PAX5 P80R mutation (marked as “PAX5 P80R”) is shown between copy number of PAX5 (marked as “PAX5 CNA”) and detailed PAX5 mutations (marked as “PAX5 (44)”, indicating 44 cases with PAX5 mutations) to illustrate the fact that homozygous (Homo.) PAX5 mutations result from loss of the wild-type allele of PAX5, while cases with heterozygous (Hetero.) P80R mutations are usually observed with a second hit to disrupt function of the other copy of PAX5. WT, wild type; NA, not available.

Figure 4.. Distribution of signaling mutations in B-ALL subtypes.

a, Distribution of mutations in 3 key signaling pathways in different B-ALL subtypes. Ras pathway includes sequence mutations from NRAS, KRAS and PTPN11; JAK/STAT pathway includes JAK1/2/3 and IL7R sequence mutations, JAK2/TYK2, EPOR and CRLF2 rearrangements. The mutations were called from RNA-seq data. The total sample number (N) for each subtype is indicated in parentheses. b, Distribution of frequently mutated signaling genes (according to PAX5 P80R group) in different B-ALL subtypes.

Figure 5.. Gene expression signature of PAX5 P80R.

a, Heatmap of top 100 differentially expressed genes (based on two-sided Wald test and Benjamini-Hochberg adjustment, 11 up-regulated and 89 down-regulated) in PAX5 P80R (N=33) vs. other B-ALL subtypes (N=372). Up-regulated genes in PAX5 P80R subtype are listed in the figure. For subtypes with many available samples, only 30 with top RNAseq quality (based on 30X coverage) are included. Full list of the genes in the signature is provided in Supplementary Table 17. Up- and down-regulated genes are ordered in the heatmap according to the significance of the adjusted P value. b, Venn diagram of differentially expressed genes (≥2 fold-change and two-sided Wald test and Benjamini-Hochberg adjusted P<0.01) in PAX5 P80R (N=33) and PAX5alt groups (N=85) versus other B-ALL (N=372). c, Gene set enrichment analysis (GSEA) of PAX5 P80R subtype (N=33) versus PAX5alt group (N=85). Gene set “B cells up” was derived from gene expression profiling of mouse hematopoietic lineages. False discovery rate (FDR), nominal p-value and normalized enrichment score are calculated by GSEA.

Figure 6.. Event-free (EFS) and overall survival (OS) of PAX5 P80R subtype.

a, Kaplan-Meier estimates of EFS and OS for children with B-ALL treated on COG NCI HR AALL0232 protocol (favorable subtypes includes High hyperdiploid, ETV6-RUNX1 and TCF3PBX1, 132 patients; DUX4, 28; KMT2A, 11; PAX5 P80R, 8; PAX5alt, 46; Ph, 18; Ph-like, 70; Other includes CRLF2 (non-Ph-like), ETV6-RUNX1-like, High hypodiploid, iAMP21, IKZF1 N159Y, MEF2D, NUTM1, ZNF384 and all other, 85). P values were calculated by the two-sided time-stratified Cochran–Mantel–Haenszel test across all the subtypes in each panel. b, Kaplan-Meier estimates of EFS and OS for adult B-ALL patients (>18 years) (BCL2/MYC, 7 patients; DUX4, 13; Hypodiploid, 26; KMT2A, 35; PAX5 P80R, 15; PAX5alt, 27; Ph, 31; Ph-like, 59; Other includes High hyperdiploid, CRLF2 (non-Ph-like), ETV6-RUNX1, iAMP21, IKZF1 N159Y, MEF2D, ZNF384 and all other, 75).

Figure 7.. PAX5 P80R impairs B cell differentiation and drives development of B-ALL.

a, Flow cytometric immunophenotyping of ex vivo cultures derived from Pax5^−/− lineage-negative bone marrow cells transduced with empty vector, wild-type PAX5, or point mutants within the DNA-binding domain of PAX5 (p.Pro80Arg (P80R), p.Val26Gly (V26G) and p.Pro34Gln (P34Q)). Cultures were grown on IL7-secreting supportive T220 stromal cells to promote differentiation to B220⁺ CD19⁺ pre-B cells. Each flow panel is a representation of at least three identical but independent experiments. b, Kaplan-Meier survival curve for mice harboring Pax5^P80R or Pax5^G183S point mutations; ***denotes two-sided log-rank Mantel-Cox test P <0.0001, N= 212 total mice, all weaned mice in the colony were included on study (66 Pax5^+/+, 11 Pax5+/−, 75 Pax5G183S/+, 22 Pax5G183S/G183S, 31 Pax5P80R/+, 7 Pax5P80R/P80R). c, Flow cytometric analysis of bone marrow samples from moribund Pax5^P80R/+ and Pax5^P80R/P80R mice for lineage markers B220 (B lymphocyte), CD3 (T lymphocyte), Mac 1 (monocyte) and Gr1 (granulocyte), CD41 (megakaryocyte) and Ter119 (erythrocyte) and a Hardy B cell panel (CD43, B220, CD19, BP1, IgM) to determine the immunophenotype of leukemic cells. Flow panels are representative of one mouse of each genotype out of a total of 10 Pax5^P80R/+ and three Pax5^P80R/P80R mice analyzed. d, Representative Giemsa-Wright stained bone marrow samples from moribund Pax5^P80R/+ mice; scale bars = 20μm; 17 independent Pax5^P80R/+ and 5 Pax5^P80R/P80R samples were analyzed with similar results e, Kaplan-Meier curve of secondary transplant recipient mice (N=3 mice per group). f, Array comparative genomic hybridization data for representative Pax5^P80R/+ and Pax5^P80R/P80R primary tumors, indicating focal and broad deletions/amplifications affecting the Pax5 locus. Animal IDs are in parentheses. Copy number alterations were detectable in three out of four mice analyzed. g, Immunoblot for PAX5, STAT5, and pSTAT5 in mouse fibroblasts (NIH3T3, negative control), B220+ splenocytes, and in vitro cultures of bone marrow cells collected from secondary transplant recipients (717–1, 731–1, 880–2, and 898–1). PAX5 antibodies detecting the N- or C-terminus were used to confirm a truncation observed in 731–1. ACTIN was used as a loading control. Immunoblots were repeated three times. h, Gene set enrichment analysis for the PAX5 P80R human B-ALL subtype versus normal human B cells isolated from bone marrow. Gene sets were derived from top 500 up or down regulated genes between Pax5 P80R (N=4) leukemia cells vs normal B samples (N=3) from mouse model. False discovery rate (FDR), nominal p-value and normalized enrichment score are calculated by GSEA.