CBFB MYH11 Research Articles

C-CBL Inactivation Promotes Cytokine-Driven Growth of Human CD34+ Cells and AML1-ETO Expressing Cells

Abstract 2464C-CBL is an E3 ubiquitin ligase and negatively regulates activated receptor tyrosine kinases. C-CBL mutations have been frequently found in a variety of myeloid neoplasms, most frequently in chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML). The mutations occur almost exclusively in exons 8–9 that encode linker/RING finger domains, which result in loss of E3 ligase activity. These mutants are considered to have dominant-negative function over the wild-type C-CBL. In acute myeloid leukemia (AML), C-CBL mutations are associated with core binding factor (CBF) leukemia. Although the physiologic roles of C-CBL and its mutations have been elucidated with several mouse models, the precise roles of C-CBL in primary human hematopoietic cells remain unclear.We have developed a unique culture system to model myeloid leukemogenesis using human CD34+ cord blood (CB) cells cultured in cytokines (SCF, TPO, Flt3 ligand, IL-3, IL-6), and have shown that CBF fusion oncoproteins, AML1-ETO (AE) and CBFB-MYH11 (CM), enable long-term proliferation in vitro by increasing self-renewal of CD34+ cells. Using this system, we assessed functions of C-CBL and its disease-related mutants in primary human CD34+ CB cells. All 3 C-CBL mutants (Gln367Pro, Tyr371Ser, DExon8/9), but not wild-type C-CBL, promoted the growth of CD34+ CB cells. However, unlike cells expressing AE or CM, C-CBL mutant-expressing cells stopped growing at about the same time as normal CB cells (3–4 weeks), suggesting that the mutants do not increase the self-renewal of CD34+ cells. Similar to the results obtained for normal CB cells, all 3 C-CBL mutants accelerated the cytokine-driven expansion of AE cells.Next, to test functions of endogenous C-CBL, we knocked down C-CBL expression in AE-expressing cells using 5 different shRNAs. To our surprise, one shRNA promoted, while the other 4 inhibited the growth of AE cells. The disparate effects of shRNAs were not due to different knockdown efficiencies of each shRNA, because the two best shRNAs exhibited nearly complete C-CBL depletion but showed either growth-promoting (shCBL-1) or growth-inhibitory (shCBL-2) effects. To determine which effect of shRNA is actually due to C-CBL downregulation, we cotransduced shRNA-resistant C-CBL together with shCBL-1 or shCBL-2 in AE cells, and compared the growth of shRNA-transduced cells and shRNA/C-CBL cotransduced cells. C-CBL reintroduction suppressed the enhanced AE cell growth by shCBL-1, while it did not rescue the defective growth of shCBL-2-transduced AE cells. These results indicate that the growth-promoting effect of shCBL-1 is actually due to C-CBL knockdown, while the growth inhibitory effect of shCBL-2 (and possibly the other 3 shRNAs) is caused by off-target and/or nonspecific toxicity of shRNAs. Transduction of the shCBL-1 also promoted the proliferation of normal CB cells. Interestingly, neither expression of C-CBL mutants nor C-CBL depletion by shCBL-1 showed growth-promoting effects on a cytokine-independent AE cell line (Kasumi-1), indicating that C-CBL inactivation promotes cell growth by enhancing the response to cytokines.To characterize the increased cell growth induced by C-CBL mutations/depletion, we assessed expression of a stem cell marker CD34 in the mutant expressing or C-CBL depleted CB and AE cells. In contrast to effects seen with CBF fusion genes, C-CBL inactivation results in decreased expression of CD34. We also performed cell cycle and apoptosis analyses, and found a significant increase in the proportion of S/G2/M phase cells upon C-CBL inactivation. These results indicate that C-CBL inactivation promotes cell growth mainly through increased cell cycle progression.In summary, we found that C-CBL mutant expression and C-CBL depletion promote cytokine-dependent growth, but do not increase self-renewal of human CD34+ CB cells (Fig.1). C-CBL mutations/depletion also showed growth-promoting effects on AE-expressing cells (Fig.1), which support the notion that C-CBL inactivation collaborates with CBF-rearranged genes (AE and CM) to promote AML. Functional cooperation between C-CBL mutations and AE in vivo is currently being tested using a xenotransplantation assay. These unique human cell models for myeloid neoplasms with C-CBL mutations/deletion will be useful to reveal their molecular pathogenesis and to find novel therapeutic targets. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Gene Mutations and Minimal Residual Disease (MRD) As Predictors of Remission Duration in Adults with Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) Treated with High-Dose Cytarabine (HDAC) - First Results of the Prospective French Intergroup CBF-2006 Trial

Abstract 410 Background.CBF-AML is a favorable AML subset cytogenetically defined by t(8;21) or inv(16)/t(16;16) rearrangements, respectively responsible for RUNX1-RUNX1T1 (CBFa) or CBFB-MYH11 (CBFb) gene fusion. Nonetheless, relapse incidence may reach 30–40% in these patients. Even if not yet prospectively validated, early MRD monitoring based on chimeric fusion gene transcript quantification by RQ-PCR may help to define patients at higher risk of relapse. Detection of activating KIT and FLT3 gene mutations has also been retrospectively associated with higher relapse rate in these patients. The French CBF-2006 trial (NCT00428558) aimed to prospectively evaluate both markers as predictors of remission duration in homogeneously treated patients. Methods.Patients (18–60y) with newly diagnosed CBF-AML were eligible. All were studied for KIT exon 8/17, FLT3-ITD, FLT3-D835, N-RAS and K-RAS mutations. They were randomized to receive timed-sequential or standard induction. Timed-sequential induction was cytarabine (AraC) 500 mg/m2 CI D1-3 and daunorubicine (DNR) 60 mg/m2 D1-3, followed by AraC 1 g/m2/12h D8-10 and DNR 35 mg/m2 D8-9. Standard arm was AraC 200 mg/m2 CI D1-7 and DNR 60 mg/m2 D1-3, followed by the second sequence at D16-18 only in case of persistent bone marrow (BM) blasts at D15. Patients in first complete remission (CR1) received 3 HDAC consolidation cycles with AraC 3 g/m2/12h D1/3/5. Fusion gene expression was quantified at baseline and before each HDAC cycle (MRD1, MRD2, MRD3) by the 100 × fusion gene/cABL ratio. Patients not reaching a 3-Log ratio reduction at MRD2 were offered allogeneic stem cell transplantation (SCT) in CR1 if they had an HLA-identical donor or could enter a single-agent dasatinib sub-study (see Boissel et al., this meeting). Results.Among the planned 200 patients randomized, 198 met eligibility criteria (96 CBFa and 102 CBFb, compared in Table 1). Overall, incidences of KIT, RAS, FLT3-D835 and FLT3-ITD mutations were 21%, 29%, 10% and 6%, respectively. Patients with KIT and FLT3-D835 mutations had higher WBC (P=0.04 and 0.02) and BM blast percentage (P=0.0004 and 0.05). Those with RAS mutations had lower BM blast percentage (P=0.01) and baseline transcript ratio (P=0.001). Overall, 196 patients reached CR1 and 55 relapsed. At 3 years, probabilities of durable remission, disease-free (DFS) and overall survival from CR were 64%, 61% and 84%, respectively, without differences between the CBF subsets or induction arms. Fifty-five patients did not reach the 3-Log MRD2 reduction (18 CBFa and 37 CBFb, P=0.006), 9 receiving SCT in CR1. Poor MRD2 response correlated with age (P=0.01), WBC (P=0.04), KIT mutations (P=0.04), and initial transcript ratio (P=0.004). Among the characteristics listed in Table 1, shorter remission duration was associated with high WBC (P=0.011), high BM blast percentage (P=0.062), del(9q) (P=0.041), KIT mutation (P=0.048), FLT3-D835 mutation (P=0.077), high initial transcript ratio (P=0.053), and <3-Log MRD2 reduction (P=0.006) in univariate analysis stratified by CBF subtype and treatment arm. In multivariate analysis, the three latter factors, but not KIT mutations, remained independently predictive of shorter remission duration and DFS (P= 0.014, 0.033, 0.002 and 0.001, 0.012, 0.005, respectively). Censoring patients receiving SCT in CR1 yielded similar results. Conclusions.This prospective study of 198 CBF-AML patients homogeneously treated with HDAC revealed significant correlations between initial characteristics and response to therapy. Prognostic analysis did not find that KIT mutations had independent impact on outcome, which was rather influenced by baseline fusion transcript ratio and post-consolidation MRD reduction (reminiscent of Schnittger et al. 2003), and FLT3-D835 mutation. Ongoing pan-genomic analyses might help to further dissect CBF-AML heterogeneity.Table 1Patient characteristicsCBFa (N = 96)CBFb (N = 102)P valuesMedian age42y42y0.37Median WBC, G/L1122<0.001Median BM blast percentage52570.10Therapy-related AML1020.016Loss of Y chromosome*330<0.001Tri 22*018<0.001Tri 8*2150.002Del(9q)*141<0.001Del(7q)/–7*450.99KIT exon 8/17/both mutation**5/16/112/6/00.38FLT3 D835/ITD/both mutation**4/6/015/4/10.11N/K-RAS mutation**13/228/110.001Median baseline transcript ratio30389<0.001>3-Log ratio reduction at MRD2***71580.006*196,**194, and***184 patients studied. Disclosures:No relevant conflicts of interest to declare.

Pro-Survival Role of RUNX1 in Acute Myeloid Leukemia with Common Fusion Proteins

Abstract 870RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Blocking RUNX1 function has been implicated in development of core-binding factor (CBF) leukemia and MLL-rearranged leukemia. In addition, inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in CBF- and MLL-rearranged leukemias, raising the possibility that a certain level of RUNX1 activity is required for efficient propagation of these leukemia cells.To determine the precise role of RUNX1 in specific types of myeloid neoplasms, we assessed RUNX1 functions in primary human CD34+ cord blood cells and those transduced with CBF related fusion oncoproteins [AML1-ETO (AE) or CBFB-MYH11 (CM)] or a MLL fusion oncoprotein, MLL-AF9 (MA9). RUNX1 was abundantly expressed and phosphorylated in AE-, CM-, and MA9-expressing long-term cultured cells. RUNX1 overexpression induced myeloid differentiation in normal CD34+ cells and prevented their long-term proliferation. Leukemogenic RUNX1 mutants lost the ability to induce differentiation, and a C-terminal truncated RUNX1 mutant conferred long-term (over 3 months) proliferative ability to CD34+ cells. RUNX1 overexpression also induced differentiation in CBF leukemia cells (AE- or CM-expressing cells). Interestingly, block of proper RUNX1 function, either by shRNA driven knockdown or forced expression of dominant-negative type mutants, showed growth inhibitory effects on CBF leukemia cells, suggesting that a certain level of RUNX1 activity is required for CBF leukemogenesis.Strikingly, block of RUNX1 function, but not RUNX1 overexpression, resulted in substantial growth inhibition of MA9 cells through enhanced apoptosis and cell cycle arrest. A xenotransplantation assay further demonstrated that RUNX1 knockdown inhibited human AML development by MA9 in vivo. The growth inhibitory effect of shRNA-mediated RUNX1 knockdown on MA9 cells was rescued by reintroduction of RUNX1, and partially restored by another RUNX transcription factor RUNX2. Thus, RUNX proteins have a growth-promoting role during MA9-driven leukemogenesis. These results contrast with those obtained using a mouse transplantation model that showed loss of Runx1 accelerates the development of MLL-ENL driven leukemia. The cause of this discrepancy is unclear, but it could be explained by species differences (human vs mouse), different experimental assays (homologous transplantation vs xenotransplantation), or the compensatory mechanism of Runx1 deletion with other Runx proteins (Runx2 and Runx3) in Runx1 knockout mice. Further studies will be needed to determine the precise roles of RUNX1 in human MLL leukemias.Finally, we assessed molecular changes in RUNX1-depleted MA9 cells and found CDKN1A upregulation and BCL2 downregulation. We also confirmed that CDKN1A depletion and BCL2 overexpression have growth-promoting effects on MA9 cells. Therefore, it appears that these molecular changes contribute to the attenuated growth of RUNX1-depleted MA9 cells. However, MA9 cells with CDKN1A depletion or BCL2 overexpression were not fully rescued from the effects of RUNX1 depletion, indicating the importance of other RUNX1 targets to support cell survival and proliferation.In conclusion, our human cell system confirmed a tumor suppressor role of RUNX1 in normal CD34+ cells, and unveiled an unexpected growth-promoting role of RUNX1 in MLL-rearranged human leukemia cells. In CBF leukemia cells, precise control of RUNX1 level appears to be important for optimal cell growth (Fig.1). Taken together, these findings suggest that partial reduction of RUNX1 activity expands myeloid progenitors by blocking differentiation, while further reduction of RUNX1 results in cell cycle arrest and increased cell death in human cells. Therefore, inhibiting RUNX1 activity rather than enhancing it will be a promising therapeutic strategy for certain types of leukemia, particularly for leukemias with common fusion proteins. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

RUNX1 repression‐independent mechanisms of leukemogenesis by fusion genes CBFB–MYH11 and AML1–ETO (RUNX1–RUNX1T1)

The core binding factor (CBF) acute myeloid leukemias (AMLs) are a prognostically distinct subgroup that includes patients with the inv(16) and t(8:21) chromosomal rearrangements. Both of these rearrangements result in the formation of fusion proteins, CBFB-MYH11 and AML1-ETO, respectively, that involve members of the CBF family of transcription factors. It has been proposed that both of these fusion proteins function primarily by dominantly repressing normal CBF transcription. However, recent reports have indicted that additional, CBF-repression independent activities may be equally important during leukemogenesis. This article will focus on these recent advances.

Acute myeloid leukemia with inv(16) with CBFB–MYH11, 3′ CBFB deletion, variant t(9;22) with BCR–ABL1, and del(7)(q22q32) in a pediatric patient: case report and literature review

Coexistence of inv(16) and t(9;22) is a rare chromosomal aberration, one that has been described in chronic myelogenous leukemia (CML), mainly in myeloid blast crisis, and de novo acute myeloid leukemia (AML). Approximately 14 cases have been reported, including only 1 pediatric case. Here we present the case of a 13-year-old boy with a new diagnosis of AML with some features of monocytic differentiation. Conventional cytogenetic analyses on unstimulated blood showed three related abnormal clones with inv(16) in the stemline: 46,XY,inv(16)(p13.1q22)[2]/46,idem,del(7)(q22q32)[16]/46,idem,t(9;22;19)(q34;q11.2;p13.1)[2]. Fluorescence in situ hybridization (FISH) studies on interphase nuclei and previously G-banded metaphases showed a 3′ CBFB deletion and confirmed the presence of the Philadelphia chromosome in a t(9;22;19) rearrangement. Deletion 7q31 was also confirmed by interphase FISH analysis. The patient was treated with standard AML chemotherapy plus gemtuzumab as part of a clinical trial. At 10-months follow-up, he was in remission. To the best of our knowledge, this is the first description of a pediatric case of de novo AML with a stemline showing inv(16) along with 3′ CBFB deletion, an abnormal clone revealing in addition a del(7)(q22q32), and another clone with a t(9;22;19)(q34;q11.2;p13.1) as an additional abnormality.

AML with CBFB–MYH11 rearrangement demonstrate RAS pathway alterations in 92% of all cases including a high frequency of NF1 deletions

AML with CBFB–MYH11 rearrangement demonstrate RAS pathway alterations in 92% of all cases including a high frequency of NF1 deletions

Banding and molecular cytogenetic studies detected a CBFB-MYH11 fusion gene that appeared as abnormal chromosomes 1 and 16 in a baby with acute myeloid leukemia FAB M4-Eo

The acute myeloid leukemia (AML) subtype M4Eo occurs in 5% of all AML cases and is usually associated with either an inv(16)(p13.1q22) or a t(16;16)(p13.1;q22) chromosomal abnormality. At the molecular level, these abnormalities generate a CBFB–MYH11 fusion gene. Patients with this genetic alteration are usually assigned to a low-risk group and thus receive standard chemotherapy. AML-M4Eo is rarely found in infants. We describe clinical, conventional banding, and molecular cytogenetic data for a 12-month-old baby with AML-M4Eo and a chimeric CBFB–MYH11 fusion gene masked by a novel rearrangement between chromosomes 1 and 16. This rearrangement characterizes a new type of inv(16)(p13.1q22) masked by a chromosome translocation.

Failure of Embryonic Hematopoiesis andLethal Hemorrhages in Mouse Embryos Heterozygousfor a Knocked-In Leukemia Gene CBFB–MYH11

The fusion oncogene CBFB–MYH11 is generated by a chromosome 16 inversion in human acute myeloid leukemia subtype M4Eo. Mouse embryonic stem (ES) cells heterozygous for this oncogene were generated by inserting part of the human MYH11 cDNA into the mouse Cbfb gene through homologous recombination (knock-in). Chimeric mice were leukemia free, but the ES cells with the knocked-in Cbfb–MYH11 gene did not contribute to their hematopoietic tissues. Mouse embryos heterozygous for Cbfb–MYH11 lacked definitive hematopoiesis and developed multiple fatal hemorrhages around embryonic day 12.5. This phenotype is very similar to that resulting from homozygous deletions of either Cbfb or Cbfa2 (AML1), consistent with a dominant negative function of the Cbfb–MYH11 fusion oncogene. An impairment of primitive hematopoiesis was also observed, however, suggesting a possible additional function of Cbfb–MYH11.