Cihangir Duy

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.

The B cell mutator AID promotes B lymphoid blast crisis and drug resistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is induced by BCR-ABL1 and can be effectively treated for many years with Imatinib until leukemia cells acquire drug resistance through BCR-ABL1 mutations and progress into fatal B lymphoid blast crisis (LBC). Despite its clinical significance, the mechanism of progression into LBC is unknown. Here, we show that LBC but not CML cells express the B cell-specific mutator enzyme AID. We demonstrate that AID expression in CML cells promotes overall genetic instability by hypermutation of tumor suppressor and DNA repair genes. Importantly, our data uncover a causative role of AID activity in the acquisition of BCR-ABL1 mutations leading to Imatinib resistance, thus providing a rationale for the rapid development of drug resistance and blast crisis progression.

Pre–B cell receptor–mediated cell cycle arrest in Philadelphia chromosome–positive acute lymphoblastic leukemia requires IKAROS function

B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre–B cell receptor–dependent stages. The Philadelphia chromosome–positive (Ph+) subtype of ALL accounts for 25–30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre–B cell receptor functions as a tumor suppressor upstream of IKAROS through induction of cell cycle arrest in Ph+ ALL cells. Pre–B cell receptor–mediated cell cycle arrest in Ph+ ALL cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre–B cell receptor signaling pathway, even if expression of the pre–B cell receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre–B cell receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph+ ALL and identify IKAROS-mediated cell cycle exit as the endpoint of an emerging pathway of pre–B cell receptor–mediated tumor suppression.

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.

Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1–transformed acute lymphoblastic leukemia cells

The Philadelphia chromosome (Ph) encoding the oncogenic BCR-ABL1 kinase defines a subset of acute lymphoblastic leukemia (ALL) with a particularly unfavorable prognosis. ALL cells are derived from B cell precursors in most cases and typically carry rearranged immunoglobulin heavy chain (IGH) variable (V) region genes devoid of somatic mutations. Somatic hypermutation is restricted to mature germinal center B cells and depends on activation-induced cytidine deaminase (AID). Studying AID expression in 108 cases of ALL, we detected AID mRNA in 24 of 28 Ph+ ALLs as compared with 6 of 80 Ph− ALLs. Forced expression of BCR-ABL1 in Ph− ALL cells and inhibition of the BCR-ABL1 kinase showed that aberrant expression of AID depends on BCR-ABL1 kinase activity. Consistent with aberrant AID expression in Ph+ ALL, IGH V region genes and BCL6 were mutated in many Ph+ but unmutated in most Ph− cases. In addition, AID introduced DNA single-strand breaks within the tumor suppressor gene CDKN2B in Ph+ ALL cells, which was sensitive to BCR-ABL1 kinase inhibition and silencing of AID expression by RNA interference. These findings identify AID as a BCR-ABL1–induced mutator in Ph+ ALL cells, which may be relevant with respect to the particularly unfavorable prognosis of this leukemia subset.

Hematopoietic Stem Cell Origin of BRAFV600E Mutations in Hairy Cell Leukemia

The cell of origin for the chronic lymphoproliferative disorder hairy cell leukemia is a long-term hematopoietic stem cell, as shown through human genetic data and murine genetic models. Finding the Origin Story for a Leukemia The cells that give rise to a cancer called hairy cell leukemia are hematopoietic stem cells, the precursors for all the types of normal blood cells, according to a new study by Chung et al. Although hairy cell leukemia is usually thought to be derived from mature B cells, it has not previously been matched with any specific stage of normal B cell development. Now, the authors performed detailed genetic analysis of human leukemia samples and also modeled this cancer in mice with different types of mutations, thus revealing the origin for hairy cell leukemia. Understanding the causes of this leukemia should help guide the design of effective treatments and may improve our understanding of similar cancers. Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder characterized by somatic BRAFV600E mutations. The malignant cell in HCL has immunophenotypic features of a mature B cell, but no normal counterpart along the continuum of developing B lymphocytes has been delineated as the cell of origin. We find that the BRAFV600E mutation is present in hematopoietic stem cells (HSCs) in HCL patients, and that these patients exhibit marked alterations in hematopoietic stem/progenitor cell (HSPC) frequencies. Quantitative sequencing analysis revealed a mean BRAFV600E-mutant allele frequency of 4.97% in HSCs from HCL patients. Moreover, transplantation of BRAFV600E-mutant HSCs from an HCL patient into immunodeficient mice resulted in stable engraftment of BRAFV600E-mutant human hematopoietic cells, revealing the functional self-renewal capacity of HCL HSCs. Consistent with the human genetic data, expression of BRafV600E in murine HSPCs resulted in a lethal hematopoietic disorder characterized by splenomegaly, anemia, thrombocytopenia, increased circulating soluble CD25, and increased clonogenic capacity of B lineage cells—all classic features of human HCL. In contrast, restricting expression of BRafV600E to the mature B cell compartment did not result in disease. Treatment of HCL patients with vemurafenib, an inhibitor of mutated BRAF, resulted in normalization of HSPC frequencies and increased myeloid and erythroid output from HSPCs. These findings link the pathogenesis of HCL to somatic mutations that arise in HSPCs and further suggest that chronic lymphoid malignancies may be initiated by aberrant HSCs.

BACH2–BCL6 balance regulates selection at the pre-B cell receptor checkpoint

At the pre-B cell receptor (BCR) checkpoint, developing pre-B cells are selected for successful rearrangement of V(H)-DJ(H) gene segments and expression of a pre-BCR. Reduced stringency at this checkpoint may obstruct the B cell repertoire with nonfunctional B cell clones. Earlier studies have described that activation of B cell lymphoma/leukemia (BCL)6 by a functional pre-BCR mediates positive selection of pre-B cells that have passed the checkpoint. This concept is now further elaborated by the recent finding that the BTB and CNC homology 1 basic leucine zipper transcription factor 2 (BACH2) induces negative selection and opposes BCL6 function prior to the pre-BCR checkpoint. Here, we discuss the antagonism between BCL6 and BACH2 during early B cell development, as well as its implications in both repertoire selection and counter-selection of premalignant clones for leukemia suppression.

Abstract LB-235: BCL6 enables Ph+ acute lymphoblastic leukemia cells to survive BCR-ABL1 kinase inhibition

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Tyrosine kinase inhibitors (TKI) are widely used to treat patients with leukemia driven by BCR-ABL1 and other oncogenic tyrosine kinases. Recent efforts focused on the development of more potent TKI that also inhibit mutant tyrosine kinases. However, even effective TKI typically fail to eradicate leukemia-initiating cells, which often cause recurrence of leukemia after initially successful treatment. Here we report on the discovery of a novel mechanism of drug-resistance, which is based on protective feedback signaling of leukemia cells in response to TKI-treatment. We identified BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukemia-initiating subclones. BCL6 is a known proto-oncogene that is often translocated in diffuse large B cell lymphoma (DLBCL). In response to TKI-treatment, BCR-ABL1 acute lymphoblastic leukemia (ALL) cells upregulate BCL6 protein levels by ∼90-fold, i.e. to similar levels as in DLBCL. Upregulation of BCL6 in response to TKI-treatment represents a novel defense mechanism, which enables leukemia cells to survive TKI-treatment: Previous work suggested that TKI-mediated cell death is largely p53-independent. Here we demonstrate that BCL6 upregulation upon TKI-treatment leads to transcriptional inactivation of the p53 pathway. BCL6-deficient leukemia cells fail to inactivate p53 and are particularly sensitive to TKI-treatment. BCL6-/-leukemia cells are poised to undergo cellular senescence and fail to initiate leukemia in serial transplant recipients. A combination of TKI-treatment and a novel BCL6 peptide inhibitor markedly increased survival of NOD/SCID mice xenografted with patient-derived BCR-ABL1 ALL cells. We propose that dual targeting of oncogenic tyrosine kinases and BCL6-dependent feedback represents a novel strategy to eradicate drug-resistant and leukemia-initiating subclones in tyrosine kinase-driven leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-235. doi:10.1158/1538-7445.AM2011-LB-235