Kadriye Nehir Cosgun

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.

PTEN opposes negative selection and enables oncogenic transformation of pre-B cells

Phosphatase and tensin homolog (PTEN) is a negative regulator of the phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) signaling pathway and a potent tumor suppressor in many types of cancer. To test a tumor suppressive role for PTEN in pre-B acute lymphoblastic leukemia (ALL), we induced Cre-mediated deletion of Pten in mouse models of pre-B ALL. In contrast to its role as a tumor suppressor in other cancers, loss of one or both alleles of Pten caused rapid cell death of pre-B ALL cells and was sufficient to clear transplant recipient mice of leukemia. Small-molecule inhibition of PTEN in human pre-B ALL cells resulted in hyperactivation of AKT, activation of the p53 tumor suppressor cell cycle checkpoint and cell death. Loss of PTEN function in pre-B ALL cells was functionally equivalent to acute activation of autoreactive pre–B cell receptor signaling, which engaged a deletional checkpoint for the removal of autoreactive B cells. We propose that targeted inhibition of PTEN and hyperactivation of AKT triggers a checkpoint for the elimination of autoreactive B cells and represents a new strategy to overcome drug resistance in human ALL.

Author Correction: Metabolic gatekeeper function of B-lymphoid transcription factors

Author(s): Chan, LN; Chen, Z; Braas, D; Lee, J-W; Xiao, G; Geng, H; Cosgun, KN; Hurtz, C; Shojaee, S; Cazzaniga, V; Schjerven, H; Ernst, T; Hochhaus, A; Kornblau, SM; Konopleva, M; Pufall, MA; Cazzaniga, G; Liu, GJ; Milne, TA; Koeffler, HP; Ross, TS; Sanchez-Garcia, I; Borkhardt, A; Yamamoto, KR; Dickins, RA; Graeber, TG; Muschen, M | Abstract: In Fig. 3c of this Letter, the the effects of CRISPR-Cas9-mediated deletion of NR3C1, TXNIP and CNR2 in patient-derived B-lineage leukaemia cells were shown. For curves depicting NR3C1 (left graph), data s for TXNIP (middle graph) were inadvertently plotted. This figure has been corrected online, and the original Fig. 3c is shown as Supplementary Information to this Amendment for transparency. The error does not affect the conclusions of the Letter. In addition, Source Data files have been added for the Figs. 1-4 and Extended Data Figs. 1-10 of the original Letter.

Abstract 93: Transcriptional control of glucocorticoid responses in leukemia

Glucocorticoids (GCs) are central to all major therapy regimens for pre-B cell-derived acute lymphoblastic leukemia (ALL), but have no activity in myeloid leukemia. Such divergent responses represent an empirically established clinical standard; however, neither the mechanism by which GCs induce cell death nor the biological basis for the distinct responses in B-cell and myeloid leukemias is clear. Studying patient-derived samples revealed that NR3C1 (glucocorticoid receptor) levels were 6- to 20-fold higher in pre-B ALL compared to chronic myeloid leukemia (CML). High levels of Nr3c1 were reduced upon B- to myeloid-lineage conversion, suggesting that regulation of NR3C1 expression and GC responsiveness depend on a B-cell transcriptional program. B-cell transcription factors (e.g. PAX5, IKZF1) are critical for B-cell development, yet they are genetically lesioned in more than 80% of pre-B ALL cases. Despite such high frequency, the significance of these inactivating lesions remains elusive. Combining ChIP-seq and RNA-seq analyses, we identified a novel B-cell transcriptional program for activation of NR3C1 and its transcriptional target TXNIP (a negative regulator of glucose uptake). Reconstitution of PAX5 or IKZF1 expression in haploinsufficient patient-derived pre-B ALL cells increased NR3C1 and TXNIP levels. Conversely, expression of dominant negative mutant of PAX5 or IKZF1 abolished NR3C1 expression. Loss of Nr3c1 or Txnip in murine BCR-ABL1-driven pre-B ALL cells resulted in survival advantage in competitive growth assays. Importantly, loss of Nr3c1 or Txnip significantly elevated glucose uptake, lactate production and cellular ATP levels. These findings suggest that GCs induce cell death by exacerbating glucose and energy depletion. Notably, reconstitution of PAX5 or IKZF1 rendered haploinsufficient patient-derived pre-B ALL cells more sensitive to dexamethasone (dex) treatment. In contrast, dominant-negative PAX5 or IKZF1 largely de-sensitized pre-B ALL cells expressing wildtype PAX5 or IKZF1. These findings suggest that B-cell transcription factors set the threshold for GC responsiveness in pre-B ALL. Since relapsed ALL cells often acquire GC resistance, drug-combinations may be useful to prevent GC-resistance. As expected, loss of Nr3c1 abrogated responses to GCs. Interestingly, loss of Txnip also largely rescued GC-induced cell death in pre-B ALL cells. On this basis, we tested drug interactions between GCs and TXNIP agonists, 3-O-methylglucose (3-OMG) and D-allose. Treating patient-derived GC-refractory pre-B ALL cells with 3-OMG or D-allose strongly synergized with GC-treatment. Collectively, our findings provide a mechanistic explanation for the empiric finding that GCs are effective in the treatment of B-cell but not myeloid malignancies, and identify TXNIP as a novel therapeutic target in pre-B ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Zhengshan Chen, Gang Xiao, Jae Woong Lee, Kadriye Nehir Cosgun, Huimin Geng, Valeria Cazzaniga, Hilde Schjerven, Ross A. Dickins, Markus Muschen. Transcriptional control of glucocorticoid responses in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 93. doi:10.1158/1538-7445.AM2017-93