ERG and c-MYC regulate a critical gene network in BCR::ABL1-driven B cell acute lymphoblastic leukemia

Abstract

Philadelphia chromosome–positive B cell acute lymphoblastic leukemia (B-ALL), characterized by the BCR::ABL1 fusion gene, remains a poor prognosis cancer needing new therapeutic approaches. Transcriptomic profiling identified up-regulation of oncogenic transcription factors ERG and c-MYC in BCR::ABL1 B-ALL with ERG and c-MYC required for BCR::ABL1 B-ALL in murine and human models. Profiling of ERG- and c-MYC–dependent gene expression and analysis of ChIP-seq data established ERG and c-MYC coordinate a regulatory network in BCR::ABL1 B-ALL that controls expression of genes involved in several biological processes. Prominent was control of ribosome biogenesis, including expression of RNA polymerase I (POL I) subunits, the importance of which was validated by inhibition of BCR::ABL1 cells by POL I inhibitors, including CX-5461, that prevents promoter recruitment and transcription initiation by POL I. Our results reveal an essential ERG- and c-MYC–dependent transcriptional network involved in regulation of metabolic and ribosome biogenesis pathways in BCR::ABL1 B-ALL, from which previously unidentified vulnerabilities and therapeutic targets may emerge.

INTRODUCTION

Acute lymphoblastic leukemia (ALL) is a highly aggressive cancer. Numerous genetic subtypes of ALL have been identified, including Philadelphia chromosome–positive B cell ALL (Ph⁺B-ALL) that comprises ~25 to 30% of all adult ALL. Ph⁺B-ALL is characterized by the t(9;22) chromosomal translocation that generates the BCR::ABL1 fusion gene resulting in abnormal tyrosine kinase signaling in B lymphoid progenitors that drives leukemia development. Ph⁺B-ALL remains a poor-prognosis cancer (1–3) despite the improved outcomes achieved by targeting the driver BCR::ABL1 fusion protein with tyrosine kinase inhibitors (TKIs) (4). More than 50 to 60% of patients relapse (5–7), including those receiving newer immunotherapeutic approaches. A better understanding of disease pathogenesis would assist rational development of new targeted therapeutic approaches.

While additional genomic lesions in BCR::ABL1 B-ALL have been identified, these are largely loss-of-function alleles, including the B cell transcription factor genes IKZF1 (IKAROS), PAX5, and EBF1, that are clinically associated with poorer disease outcomes (8–11). Loss of these factors in preclinical models has been associated with accelerated leukemia expansion that is proposed to occur via loss of their metabolic gatekeeper function (12), suggesting that these genes and members of their gene regulatory networks are unlikely to represent viable molecular candidates as direct therapeutic targeting of loss-of-function alterations is problematic.

We sought to specifically identify gain of function and thus potentially targetable alterations that contribute to Ph⁺B-ALL pathogenesis, including those that may not be identified through diagnostic genomic assays. To do this, we used a murine model of BCR::ABL1 B-ALL to identify transcription factors and gene regulatory networks required for leukemia initiation and progression. Transcriptional profiling revealed up-regulation of ERG and c-MYC in murine and human BCR::ABL1 B-ALL, and, in murine and human models, ERG and c-MYC were required for BCR::ABL1 B-ALL. Profiling of ERG- and c-MYC–dependent gene expression and analysis of chromatin immunoprecipitation sequencing (ChIP-seq) data established that ERG and c-MYC coordinate a regulatory network in BCR::ABL1 B-ALL directly controlling expression of genes enriched for involvement in several cellular processes. Prominent was control of ribosome biogenesis, including direct regulation of RNA polymerase I (POL I) subunits, the role of which was validated by inhibition of BCR::ABL1 cells and other genomic subtypes by POL I inhibitors.

RESULTS

ERG, c-MYC, and downstream gene targets are highly expressed in Ph⁺B-ALL

We initially determined the transcriptional changes associated with leukemic transformation in BCR::ABL1 B-ALL in the P190 transgenic mouse model that expresses a human BCR::ABL1 transgene under the control of the metallothionein promoter (13). This transgenic mouse develops a highly penetrant acute pre-B cell (precursor B cell) lymphoblastic leukemia that closely phenocopies human disease. The murine model is characterized by expansion of pre-B cells in the bone marrow, typically between 5 and 8 weeks of age, and a B-lineage differentiation block resulting in a deficiency of maturing immunoglobulin M (IgM)⁺IgD⁺ B cells (Fig. 1A).

Pre-B cells from leukemia-bearing P190 mice were isolated, and genome-wide gene expression profiling was undertaken by bulk RNA sequencing (RNA-seq) comparing leukemic cells to control (non-transformed) C57BL/6 pre-B cells. This analysis identified significant transcriptional deregulation of 9258 genes in P190 pre-B leukemic cells (4720 up and 4538 down; table S1). This included down-regulation of genes implicated in B-lineage differentiation, such as Ikzf1 (14, 15), Ikzf3 (Aiolos) (16), Pax5 (17, 18), Tcf3(E2A) (19, 20), and Irf4 and Irf8 (21) (fig. S1A) that may contribute to the differentiation block observed in P190 leukemic mice. Of note, loss-of-function alleles of several of these down-regulated genes have been identified in human ALL validating the use of this model (8, 9).

Comparison of transcriptional changes in murine P190 leukemic pre-B cells to publicly available gene expression data from human BCR::ABL1 B-ALL (GSE5314) (22) revealed significant correlation with transcriptional changes in human B-ALL (Fig. 1, B and C).

Examination of transcription factors from the Kyoto Encyclopedia of Genes and Genomes deregulated in cancer gene set (ko05202) identified up-regulation of several oncogenic transcription factors involved in B cell differentiation, including the E26 Transformation-Specific (ETS) family transcription factor ERG (23), the β helix–loop–helix transcription factor c-MYC (24) and its cooperative transcriptional partner MAX (25) in both murine and human BCR::ABL1 B-ALL (Fig. 1C). Increased protein levels of ERG and MYC were observed in P190 B-ALL cell lines compared to control pre-B cells (Fig. 1D).

Consistent with regulation by ERG and c-MYC of gene networks required for BCR::ABL1 B-ALL leukemogenesis, significant enrichment for the ETS, c-MYC, and MAX binding motifs was found in genes up-regulated in murine and human BCR::ABL1 B-ALL (Fig. 1E). Gene Ontology (GO) analysis of up-regulated genes in murine and human BCR::ABL1 B-ALL showed that the most significantly enriched gene sets were associated with ribosome biogenesis, as well as processes such as amide biosynthesis and nucleotide, tRNA, and amino acid metabolism (Fig. 1F and table S1). Up-regulation of ribosome biogenesis in BCR::ABL1 leukemia was confirmed by significantly increased transcription of the POL I–dependent 47S/45S precursor ribosomal RNA (rRNA) internal transcribed spacer in P190 leukemia cells compared to wild-type pre-B cells (fig. S1B).

ERG and c-MYC are critical for Ph⁺B-ALL development

To explore the functional significance of ERG and c-MYC in Ph⁺B-ALL, we first examined whether co-occurrence of BCR::ABL1 and ABL1 class fusions occurred with genomic variants of ERG and c-MYC in a cohort of human ALL (St. Jude, PeCan, accessed 21 January 2022) (26). While loss-of-function variants and copy number loss of ERG and, to a smaller extent, c-MYC were identified in this cohort of B-ALL, these did not co-occur with BCR::ABL1 or ABL1 class fusions (fig. S1C). Examination of CRISPR-Cas9–based gene dependency screening by the Cancer Dependency Map initiative (27) demonstrated that, while gene dependency for ERG and c-MYC could be variably identified for several genomic subtypes in B-ALL cell lines, no BCR::ABL1 class cell lines had been assessed (fig. S1D).

To directly address the requirement for ERG and c-MYC in BCR::ABL1 B-ALL, we generated P190 mice in which the Erg or c-Myc genes were deleted from the Common lymphoid progenitor (CLP) stage of lymphopoiesis using a Rag1-Cre conditional knockout approach (28). Deletion of a single allele of Erg (P190^T/+;Rag1Cre^T/+;Erg^∆/+) was sufficient to prevent the development of P190 B-ALL in this model. Deletion of one c-Myc allele (P190^T/+;Rag1Cre^T/+;c-Myc^Δ/+) also significantly delayed leukemia development, and this delay was more pronounced in the absence of both c-Myc alleles (P190^T/+;Rag1Cre^T/+;c-Myc^Δ/Δ) (Fig. 2A). At 5 weeks of age, pre-B cell numbers in P190 bone marrow were similar to wild-type mice, whereas, at 8 weeks of age, before overt symptoms of P190 disease, a significant proportion of P190 mice had developed an abnormal accumulation of pre-B cells (fig. S1E). In contrast, in P190 mice lacking one copy of Erg or c-Myc, no expansion of pre-B cells was seen, with pre-B cell numbers in 8-week-old P190^T/+;Rag1Cre^T/+;Erg^Δ/+ and P190^T/+;Rag1Cre^T/+;c-Myc^Δ/+ mice comparable to those seen in C57BL/6 controls (Fig. 2B). To determine the impact of loss of Erg or c-Myc alleles on clonal expansion during BCR::ABL1 leukemogenesis, immunoglobulin heavy-chain (Igh) gene clonotyping analysis was performed on bulk RNA-seq data obtained from primary pre-B cells. In P190 mice, leukaemogenesis was associated with dominant clones arising at 5 and 8 weeks of age, with one clone often becoming dominant in mice developing overt leukemia (Fig. 2C). In contrast, quantitative analysis of the 10 most frequent Igh clones revealed no significant dominant clonal expansion in P190 ^T/+;Rag1Cre^T/+;Erg^Δ/+ mice at 8 weeks of age. Similarly, dominant clonal expansion was not observed in P190^T/+;Rag1Cre^T/+;c-Myc^Δ/+ mice. Together, these data demonstrate high expression of ERG and c-MYC in BCR::ABL1 B-ALL compared to non-transformed pre-B cells and that these transcription factors are necessary for pre-B cell clonal expansion and subsequent leukemia development.

ERG and c-MYC contribute to BCR::ABL1 B-ALL maintenance

We next assessed the role of ERG and c-MYC in sustaining established BCR::ABL1 leukemia. We derived multiple independent cell lines from leukemic P190 mice carrying either floxed Erg (Erg^fl/fl) or c-Myc (c-Myc^fl/fl) alleles in addition to the CreERT2 transgene (29) to allow 4-hydroxy-tamoxifen (4-OHT)–dependent deletion of the floxed alleles (Fig. 3A, top). Upon 4-OHT treatment, reduced expression of ERG or c–MYC was observed in the respective cell lines, in which Erg or c-Myc was conditionally deleted (Fig. 3B). Dose-responsive inhibition of these ERG and c-MYC–deficient leukemic cell lines was observed, an effect not seen in in P190^T/+;CreER^T/+ control leukemia cells (Fig. 3C).

We next investigated the requirement for ERG and c-MYC in BCR::ABL1 leukemia maintenance in vivo. Individual P190^T/+;CreER^T/+;Erg^fl/fl and P190^T/+;CreER^T/+;c-Myc^fl/fl leukemia cell lines were pretreated with either 4-OHT (pre4-OHT) resulting in significant loss of Erg or c-Myc expression (Fig. 3D) or vehicle control and then transplanted into irradiation-conditioned recipients. Mice that received vehicle-treated cells were then either given tamoxifen (TAM) at day 8 or left untreated (control group) (Fig. 3A, bottom). In recipients of cells that are ERG- or MYC-deficient, by either 4-OHT pretreatment or in vivo TAM administration, the time to ethical endpoint due to leukemia was prolonged relative to control mice (Fig. 3E). Splenomegaly was observed to be significantly reduced in cohorts that received c-MYC–deficient cells (Fig. 3F), while the proportion of donor cells in bone marrow was observed to be consistently high in all groups (Fig. 3G). Notably, leukemia that developed in mice transplanted with P190^T/+;CreER^T/+;Erg^fl/fl and P190^T/+;CreER^T/+;c-Myc^fl/fl cell lines following 4-OHT treatment or TAM treatment in vivo demonstrated incomplete and variable reduction in ERG or c-MYC expression in the diseased bone marrow cells (fig. S2A), suggesting in vivo expansion of leukemia cells that had escaped efficient Cre-mediated gene recombination had occurred.

To confirm and extend our observations to human BCR::ABL1 B-ALL cells, guide RNAs directed against human ERG or c-MYC were expressed via a doxycycline (Dox)–inducible lentiviral vector co-expressing green fluorescent protein, resulting in reduced ERG or c-MYC protein levels in respective Cas9-expressing human BV173 cells (Fig. 4A). Reduction of ERG or c-MYC expression by two independent guide RNAs for each gene resulted in a distinct competitive proliferative disadvantage in BV173 cells compared to empty vector controls (Fig. 4B). Similar results were obtained in a second BCR::ABL1 B-ALL human cell line, SupB15 (fig. S2, B and C). Last, reduction of ERG or c-MYC either before transplantation (preDox) or treatment with Dox in vivo resulted in delayed BV173 tumor growth in transplanted mice compared to untreated controls or mice transplanted with empty vector expressing BV173 cells (control) (Fig. 4C).

In addition to BCR::ABL1 B-ALL, prominent ERG and c-MYC expression is also found in other B-ALL subtypes (fig. S3A) and pre-B-ALL human cell lines, including Nalm6 and RS4:11 (fig. S3B), where binding of ERG and c-MYC to the POLR1B promoter can be observed (fig. S3C). As observed in BCR::ABL1 B-ALL cell lines, genetic reduction of ERG or c-MYC expression in Nalm6 and RS4:11 cells resulted in a competitive proliferative disadvantage compared to empty vector controls (fig. S3, D to G).

Identification of a gene network regulated by ERG and c-MYC in BCR::ABL1 B-ALL

To define the gene networks regulated by ERG and c-MYC that facilitate leukaemogenesis, we first examined the gene expression changes upon deletion of either Erg or c-Myc in established P190^T/+;CreERT2^T/+;Erg^fl/fl and P190^T/+;CreERT2^T/+;c-Myc^fl/fl cell lines (table S2). Notably, there was overlap of differentially expressed genes upon Erg or c-Myc deletion in genetically independent cell lines. Of particular interest were genes down-regulated with both Erg or c-Myc deletion (Fig. 5A), as these genes may form part of a transcriptional gene network regulated by ERG and c-MYC, mediating the functional roles for these transcription factors in BCR::ABL1 B-ALL during leukaemogenesis and in leukemia maintenance...