Identification of a recurrent germline PAX5 mutation and susceptibility to pre-B cell acute lymphoblastic leukemia

Somatic alterations of the lymphoid transcription factor gene PAX5 are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL)^1-3, but inherited mutations of PAX5 have not previously been described. Here, we report a novel heterozygous germline variant, c.547G>A (p.Gly183Ser), in the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all patients in both families exhibited 9p deletion, with loss-of-heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 Gly183 substitutions. Functional and gene expression analysis of the PAX5 mutation demonstrated relatively reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B ALL, and implicate PAX5 in a novel syndrome of susceptibility to pre-B cell neoplasia.

B-cell precursor acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. There is a 2-4 fold increased risk of developing the disease in children of affected siblings⁴, and in occasional cases ALL is inherited as a Mendelian disorder⁵. PAX5, encoding the B-cell lineage transcription factor paired box 5, is somatically deleted, rearranged or otherwise mutated in approximately 30% of sporadic B-progenitor ALL cases^1,3,6-9. In PAX5-deficient mice, B-cell development is arrested at the pro-B-cell stage, and in vitro these cells can differentiate into other lymphoid and myeloid lineages¹⁰. PAX5 is also essential for maintaining the identity and function of mature B-cells¹¹, and its deletion in mature B cells results in dedifferentiation to pro-B cells and aggressive lymphomagenesis¹².

A heterozygous germline PAX5 variant, c.547G>A (NM_016734), p.Gly183Ser (NP_057953), was identified by exome sequencing in two families, one of Puerto Rican ancestry (family 1; Figure 1a) and the other of African-American ancestry (family 2; Figure 1b, Supplementary Note). This variant had not been previously described in public databases (Exome Variant Server, 1000 Genomes and dbSNP 137), or previous sequencing analyses of ALL and cancer genomes^1,2,9. All affected family members had B-cell precursor ALL and all available diagnostic and relapse leukemic samples from both families demonstrated loss of 9p through i(9)(q10) or dicentric chromosomes involving 9q, both of which resulted in loss of the wild-type PAX5 allele and retention of PAX5 p.Gly183Ser (Figure 1c, Supplementary Figure 1 and Supplementary Table 1).

The germline PAX5 p.Gly183Ser mutation segregated with the leukemia in both kindreds, however, several unaffected obligate carriers (Family 1: II3, III2, III3 and Family 2: I1, I2, II2, II3) were also observed, suggesting incomplete penetrance. Unaffected mutation carriers and patients at the time of ALL diagnosis had normal immunoglobulin levels and no laboratory or clinical evidence of impaired B-cell function. Sanger sequencing of cDNA from peripheral blood of unaffected carriers indicated biallelic transcription of PAX5 (data not shown). The only mutated gene common to both families was PAX5 and no germline copy number aberrations were found to be shared between patients (Supplementary tables 2 and 3).

To determine whether p.Gly183Ser arose independently in each kindred or instead reflects common ancestry, the risk haplotypes of each family were compared. The families share a 4.7kb haplotype, spanning five SNPs (Figure 1d, Supplementary Note). The relatively small size of the shared haplotype and principal component analysis of genome-wide single nucleotide polymorphism genotype data (Supplementary Figure 2) together imply that the two families are not recently related and differ in ethnicity. Moreover, given the reduced fitness due to increased susceptibility for childhood ALL, it is unlikely that such a lethal mutation could be propagated over time. Because this haplotype is relatively frequent worldwide (Supplementary Table 4), it is likely that each family's mutation arose independently.

Genomic profiling of tumor samples demonstrated expression of the p.Gly183Ser mutant PAX5 in diagnostic and relapse tumor specimens from affected members of family 2, an average of 1 chimeric fusion and 9 non-silent sequence variants per case, and homozygous deletion of CDKN2A/CDKN2B in all cases due to loss of 9p and focal deletion of the second allele. Apart from loss of 9p, no other somatic sequence mutations or structural rearrangements were shared by the affected family members (Supplementary Tables 1, 5-12)

As somatic i(9)(q10)/dic(9;v) abnormalities were seen in all of the familial leukemias, we sequenced PAX5 in 44 additional sporadic pre-B ALL cases with i(9)(q10)/dic(9;v) aberrations to assess whether PAX5 mutations frequently co-occur with loss of 9p. Two leukemic samples revealed octapeptide mutations p.Gly183Ser, and p.Gly183Val, and in others previously reported variants including p.Pro80Arg and p.Val26Gly¹ were observed (Table 1, Figure 2a). We examined the frequency of non-silent PAX5 somatic sequence mutations in a cohort of B-ALL cases with i(9)(q10)/dic(9;v) (n=28) and two cohorts of B-ALL without i(9)(q10)/dic(9;v) (n=183) and n=221.^1,2. We observed a significantly higher frequency of PAX5 mutations in the i(9)/dic(9) cohort (p=0.0001). No novel germline PAX5 mutations were detected in 39 families with a history of two or more cases of cancer including at least one hematological cancer, although one familial case of ALL harbored a dic(9;20)(p11;q11.1) and a somatic p.Pro80Arg variant (Table 1 and Supplementary Materials).

Previously identified PAX5 somatic mutations commonly result in significant reduction in transcriptional activation mediated by PAX5. Downstream targets of PAX5 include CD19 and CD79A (Igα or mb-1)¹³. We examined the transactivating activity of wild type and mutant PAX5 alleles using a PAX5-dependent reporter gene assay, containing copies of a high-affinity PAX5-binding site derived from the CD19 promoter¹⁴. Both p.Gly183Ser and Val mutations resulted in partial but significant reduction in transcriptional activation (P<0.0001 for both alleles, Figure 3a). Additionally, there was no detectable differences in subcellular localization of wild type and p.Gly183Ser PAX5 (Supplementary Figure 3). To study the effect of this mutation on CD79A expression, we expressed mutant and wild type PAX5 in J558LμM, a mouse plasmacytoma cell line that does not express PAX5 or CD79A. Enforced expression of PAX5 results in expression of CD79A and assembly of the surface immunoglobulin (sIgM) complex. The amount of sIgM expression may be used to assess the transcriptional activity of PAX5 alleles on the CD79A promoter¹. Both p.Gly183 alleles resulted in a significant reduction in sIgM expression compared to wild-type PAX5 (P<0.0001, Figure 3b). These results suggest that the PAX5 G183 mutations result in partial loss of PAX5 activity.

The identified missense variant, p.Gly183Ser, is located at a conserved residue in the octapeptide (OP) domain of PAX5 that mediates interaction with Groucho transcriptional corepressors¹⁵ (Figure 2b). Previous studies have shown that GRG4 (also known as TLE4 in humans) represses PAX5-dependent luciferase activity in cells expressing wild type PAX5 but not in cells expressing PAX5 octapeptide domain mutants¹⁵. We observed GRG4-mediated repression of the transcriptional activity of PAX5 wild-type and p.Gly183Ser (Figure 3c), suggesting the effect of this mutation is not mediated by an altered interaction with GRG4.

To further explore the effect of the variant on downstream targets, we performed genome-wide transcriptional profiling of J558LμM cells transduced with empty vector, PAX5 wild-type or mutant alleles (either all transduced cells marked by RFP expression, or the subset of cells expressing sIgM), and examined the expression of PAX5 activated and repressed genes previously defined in Pax5^−/− murine pro- and mature B cells^16-19 and in human ETV6-RUNX1 B-ALL¹. Examining all PAX5 expressing cells, we observed profound deregulation of PAX5 activated and repressed genes in J558LμM cells expressing known loss of function (e.g. the common exon 2-6 deletion that results in a truncating frameshift PAX5 allele) or strongly hypomorphic alleles (PAX5 p.Pro80Arg) and less marked deregulation in p.Gly183Ser or Val expressing cells (P values for each mutant allele versus wild-type were all P<0.001 (Supplementary Figures 4 and 5). Comparing sorted sIgM positive PAX5 p.Gly183Ser to PAX5 wild-type cells, we observed reduced expression of genes activated by PAX5 in pro-B and mature B-cells (P =1.4 × 10⁻⁴ and P =3.8 × 10⁻⁴ respectively, Supplementary Tables 13-15)

We next examined the transcriptional consequences of PAX5 p.Gly183Ser by performing transcriptome sequencing (mRNA-seq) of diagnosis and relapse samples obtained from two patients in kindred 2, and of 139 sporadic childhood B-progenitor ALL samples. We performed gene set enrichment analysis incorporating gene sets of PAX5-mutated ETV6-RUNX1 ALL (one third of which harbor focal PAX5 deletions)¹, PAX5 regulated genes in Pax5^−/− mice^16-19 and genes regulated during murine B lymphoid development²⁰. As a limited set of genes are known to be regulated in both murine pro- and mature B cells, and as the overlap between mouse and human PAX5 regulated genes are unknown, we used all previously published PAX5 regulated genes, and genes regulated during murine B cell development ^16-20 in an unbiased approach to explore the effects of the PAX5 p.Gly183 mutations on direct and indirect transcriptional targets of PAX5. This showed striking enrichment of genes deregulated in ETV6-RUNX1/PAX5-mutated ALL, PAX5 activated and repressed genes (including CD19, CD72 and CD79a), and genes regulated during murine B lymphoid development in the signature of familial PAX5 p.Gly183Ser versus sporadic B-ALL (Figure 3d and Supplementary Figures 6 and 7. We also analyzed the overlap of previously published data and the expression differences between the familial ALL tumor samples and other B-ALL cases stratified by PAX5 mutation status (Supplementary file 6; Supplementary Figure 8). Together, these results suggest that PAX5 p.Gly183Ser results in attenuation of PAX5 function and deregulation of PAX5 target genes that is less severe than the previously reported p.Pro80Arg and D2-6 alleles that result in marked or complete loss of PAX5 activity.

The PAX5 deletions, translocations and sequence mutations identified as somatic events in B-ALL commonly affect the DNA-binding and transactivation domains and result in complete loss or marked attenuation of PAX5 transcriptional activity, but are rarely homozygous and not observed as inherited variants. Moreover, Pax5 loss promotes the development of B-ALL in experimental models which are commonly accompanied by the acquisition of second hits in Pax5 ²¹, indicating that profound loss of PAX5 activity is commonly a central event in leukemogenesis. In contrast, the inherited PAX5 p.Gly183Ser mutation results in modest attenuation of PAX5 activity , and is accompanied by somatic loss of the wild-type PAX5 allele due to 9p alterations during leukemogenesis. This model is also consistent with the finding of a significant association of somatic PAX5 hypomorphic mutations coincident with complete loss of the normal PAX5 allele in leukemic cells absent 9p. These observations suggest that a severe reduction in PAX5 activity is incompatible with normal B lymphoid development and is deleterious in carriers, but by contrast, the partial hypomorphic p.Gly183Ser allele is tolerated as a germline allele but additional genetic events further reducing PAX5 activity are required to establish the leukemic clone. The universal finding of deletion of wild-type PAX5 in all familial ALL cases, rather than the acquisition of additional hypomorphic PAX5 mutations, suggests that a complete loss of wild-type PAX5 activity is required for developmental arrest and loss of maturation. This is supported by our transcriptional profiling of J558LμM p.Gly183Ser cells and familial leukemias showing deregulation of PAX5 target gene expression that is significant but less marked than known loss-of-function mutations. The differences in transcriptional profiles of some target gene panels were not as robustly observed as in mouse model systems, presumably due to inherent germline and somatic genetic and epigenetic variability in human leukemias. In addition, ongoing studies will be of interest to fully characterize the functional consequences of PAX5 octapeptide domain mutations.

Our findings have clinical implications with regard to options for pre-implantation genetic diagnosis, and the possible significance of somatic 9p alterations as a harbinger of a germline PAX5 mutation. The recent identification of germline TP53 mutations in familial ALL^20,22 and the data presented here strongly implicating PAX5 mutations in a novel syndrome of inherited susceptibility to pre-B cell ALL, indicate that further sequencing of affected kindreds is required to define the full spectrum of germline variations contributing to ALL pathogenesis.