Christian Hurtz

BCL6 enables Ph + acute lymphoblastic leukaemia cells to survive BCR–ABL1 kinase inhibition

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR–ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.

BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is induced by the oncogenic BCR-ABL1 tyrosine kinase and can be effectively treated for many years with tyrosine kinase inhibitors (TKIs). However, unless CML patients receive life-long TKI treatment, leukemia will eventually recur; this is attributed to the failure of TKI treatment to eradicate leukemia-initiating cells (LICs). Recent work demonstrated that FoxO factors are critical for maintenance of CML-initiating cells; however, the mechanism of FoxO-dependent leukemia initiation remained elusive. Here, we identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. BCL6 represses Arf and p53 in CML cells and is required for colony formation and initiation of leukemia. Importantly, peptide inhibition of BCL6 in human CML cells compromises colony formation and leukemia initiation in transplant recipients and selectively eradicates CD34+ CD38− LICs in patient-derived CML samples. These findings suggest that pharmacological inhibition of BCL6 may represent a novel strategy to eradicate LICs in CML. Clinical validation of this concept could limit the duration of TKI treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation.

BCL6 is critical for the development of a diverse primary B cell repertoire

BCL6 protects germinal center (GC) B cells against DNA damage–induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7–dependent B cell precursors, we report that IL-7Rα–Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a μ heavy chain, however, activation of pre–B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Rα–Stat5 signaling is attenuated. At the transition from IL-7–dependent to –independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre–B cells from DNA damage–induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre–B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.

BACH2 mediates negative selection and p53-dependent tumor suppression at the pre-B cell receptor checkpoint

The B cell–specific transcription factor BACH2 is required for affinity maturation of B cells. Here we show that Bach2-mediated activation of p53 is required for stringent elimination of pre-B cells that failed to productively rearrange immunoglobulin VH-DJH gene segments. After productive VH-DJH gene rearrangement, pre-B cell receptor signaling ends BACH2-mediated negative selection through B cell lymphoma 6 (BCL6)-mediated repression of p53. In patients with pre-B acute lymphoblastic leukemia, the BACH2-mediated checkpoint control is compromised by deletions, rare somatic mutations and loss of its upstream activator, PAX5. Low levels of BACH2 expression in these patients represent a strong independent predictor of poor clinical outcome. In this study, we demonstrate that Bach2+/+ pre-B cells resist leukemic transformation by Myc through Bach2-dependent upregulation of p53 and do not initiate fatal leukemia in transplant-recipient mice. Chromatin immunoprecipitation sequencing and gene expression analyses carried out by us revealed that BACH2 competes with BCL6 for promoter binding and reverses BCL6-mediated repression of p53 and other cell cycle checkpoint–control genes. These findings identify BACH2 as a crucial mediator of negative selection at the pre-B cell receptor checkpoint and a safeguard against leukemogenesis.

Self-Enforcing Feedback Activation between BCL6 and Pre-B Cell Receptor Signaling Defines a Distinct Subtype of Acute Lymphoblastic Leukemia

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.

Integrative Epigenomic Analysis Identifies Biomarkers and Therapeutic Targets in Adult B-Acute Lymphoblastic Leukemia

UNLABELLED Genetic lesions such as BCR-ABL1, E2A-PBX1, and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point toward disease mechanisms and useful biomarkers and therapeutic targets. We therefore conducted DNA methylation and gene expression profiling on a cohort of 215 adult patients with B-ALL enrolled in a single phase III clinical trial (ECOG E2993) and normal control B cells. In BCR-ABL1-positive B-ALLs, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in patients with ALL regardless of BCR-ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALLs. In E2A-PBX1-positive B-ALLs, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A-PBX1 chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq), suggesting that E2A-PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation, and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6-targeted therapy as a new therapeutic strategy for MLLr B-ALLs. SIGNIFICANCE We conducted the first integrative epigenomic study in adult B-ALLs, as a correlative study to the ECOG E2993 phase III clinical trial. This study links for the first time the direct actions of oncogenic fusion proteins with disruption of epigenetic regulation mediated by cytosine methylation. We identify a novel clinically actionable biomarker in B-ALLs: IL2RA (CD25), which is linked with BCR-ABL1 and an inflammatory signaling network associated with chemotherapy resistance. We show that BCL6 is a novel MLL fusion protein target that is required to maintain the proliferation and survival of primary human adult MLLr cells and provide the basis for a clinical trial with BCL6 inhibitors for patients with MLLr.

Mechanistic rationale for targeting the unfolded protein response in pre-B acute lymphoblastic leukemia

Significance The unfolded protein response (UPR) mitigates endoplasmic reticulum (ER) stress. In this regard, ER stress-inducing agents were found to be highly active in a clinical trial for children with relapsed acute lymphoblastic leukemia (ALL), a disease derived from transformed pre-B cells. To understand the efficacy of ER stress-inducing agents in pre-B ALL, we studied the relevance of the UPR pathway in genetic and patient-derived (xenograft) models of human pre-B ALL. Our studies revealed an unrecognized vulnerability of both normal pre-B cells and pre-B cell-derived ALL cells to genetic or pharmacological blockade of the UPR pathway. Our results establish a mechanistic rationale for the treatment of children with pre-B ALL with agents that block the UPR pathway and induce ER stress. The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the endoplasmic reticulum (ER), regulates the expression and activity of molecules including BiP (HSPA5), IRE1 (ERN1), Blimp-1 (PRDM1), and X-box binding protein 1 (XBP1). These molecules are required for terminal differentiation of B cells into plasma cells and expressed at high levels in plasma cell-derived multiple myeloma. Although these molecules have no known role at early stages of B-cell development, here we show that their expression transiently peaks at the pre–B-cell receptor checkpoint. Inducible, Cre-mediated deletion of Hspa5, Prdm1, and Xbp1 consistently induces cellular stress and cell death in normal pre-B cells and in pre–B-cell acute lymphoblastic leukemia (ALL) driven by BCR-ABL1- and NRASG12D oncogenes. Mechanistically, expression and activity of the UPR downstream effector XBP1 is regulated positively by STAT5 and negatively by the B-cell–specific transcriptional repressors BACH2 and BCL6. In two clinical trials for children and adults with ALL, high XBP1 mRNA levels at the time of diagnosis predicted poor outcome. A small molecule inhibitor of ERN1-mediated XBP1 activation induced selective cell death of patient-derived pre-B ALL cells in vitro and significantly prolonged survival of transplant recipient mice in vivo. Collectively, these studies reveal that pre-B ALL cells are uniquely vulnerable to ER stress and identify the UPR pathway and its downstream effector XBP1 as novel therapeutic targets to overcome drug resistance in pre-B ALL.

Metabolic gatekeeper function of B-lymphoid transcription factors

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.

SOX4 enables oncogenic survival signals in acute lymphoblastic leukemia

The Sox4 transcription factor mediates early B-cell differentiation. Compared with normal pre-B cells, SOX4 promoter regions in Ph(+) ALL cells are significantly hypomethylated. Loss and gain-of-function experiments identified Sox4 as a critical activator of PI3K/AKT and MAPK signaling in ALL cells. ChIP experiments confirmed that SOX4 binds to and transcriptionally activates promoters of multiple components within the PI3K/AKT and MAPK signaling pathways. Cre-mediated deletion of Sox4 had little effect on normal pre-B cells but compromised proliferation and viability of leukemia cells, which was rescued by BCL2L1 and constitutively active AKT and p110 PI3K. Consistent with these findings, high levels of SOX4 expression in ALL cells at the time of diagnosis predicted poor outcome in a pediatric clinical trial (COG P9906). Collectively, these studies identify SOX4 as a central mediator of oncogenic PI3K/AKT and MAPK signaling in ALL.

Pre-BCR signaling in precursor B-cell acute lymphoblastic leukemia regulates PI3K/AKT, FOXO1 and MYC, and can be targeted by SYK inhibition

Precursor-B-cell receptor (pre-BCR) signaling and spleen tyrosine kinase (SYK) recently were introduced as therapeutic targets for patients with B-cell acute lymphoblastic leukemia (B-ALL), but the importance of this pathway in B-ALL subsets and mechanism of downstream signaling have not fully been elucidated. Here, we provide new detailed insight into the mechanism of pre-BCR signaling in B-ALL. We compared the effects of pharmacological and genetic disruption of pre-BCR signaling in vitro and in mouse models for B-ALL, demonstrating exquisite dependency of pre-BCR+ B-ALL, but not other B-ALL subsets, on this signaling pathway. We demonstrate that SYK, PI3K/AKT, FOXO1 and MYC are important downstream mediators of pre-BCR signaling in B-ALL. Furthermore, we define a characteristic immune phenotype and gene expression signature of pre-BCR+ ALL to distinguish them from other B-ALL subsets. These data provide comprehensive new insight into pre-BCR signaling in B-ALL and corroborate pre-BCR signaling and SYK as promising new therapeutic targets in pre-BCR+ B-ALL.