Combining standard cytotoxic chemotherapy with BCR-ABL1 tyrosine kinase inhibitors (TKI) has greatly improved the upfront treatment of patients with Philadelphia chromosome-positive (Ph+) acute ...lymphoblastic leukemia (ALL). However, due to the development of drug resistance through both BCR-ABL1-dependent and -independent mechanisms, prognosis remains poor. The STAT5 transcription factor is activated by BCR-ABL1 and by JAK2-dependent cytokine signaling; therefore, inhibiting its activity could address both mechanisms of resistance in Ph+ ALL. We show here that genetic and pharmacologic inhibition of STAT5 activity suppresses cell growth, induces apoptosis, and inhibits leukemogenesis of Ph+ cell lines and patient-derived newly diagnosed and relapsed/TKI-resistant Ph+ ALL cells
and in mouse models. STAT5 silencing decreased expression of the growth-promoting PIM-1 kinase, the apoptosis inhibitors MCL1 and BCL2, and increased expression of proapoptotic BIM protein. The resulting apoptosis of STAT5-silenced Ph+ BV173 cells was rescued by silencing of BIM or restoration of BCL2 expression. Treatment of Ph+ ALL cells, including samples from relapsed/refractory patients, with the PIM kinase inhibitor AZD1208 and/or the BCL2 family antagonist Sabutoclax markedly suppressed cell growth and leukemogenesis
and in mice. Together, these studies indicate that targeting STAT5 or STAT5-regulated pathways may provide a new approach for therapy development in Ph+ ALL, especially the relapsed/TKI-resistant disease.
Suppression of STAT5 by BCL2 and PIM kinase inhibitors reduces leukemia burden in mice and constitutes a new potential therapeutic approach against Ph+ ALL, especially in tyrosine kinase inhibitor-resistant disease.
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Acute erythroleukemia (AEL or acute myeloid leukemia AML-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and ...mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.
•Transcriptomes cluster most AEL apart from other myeloid malignancies.•Alterations of AEL erythroid master regulators impair GATA1 activity and induce the disease in mice.
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Background The prognosis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has improved with minimal residual disease (MRD)-stratified therapy, however, gamma delta T cell receptor positive ...(γδ) T-ALL remains a high-risk (HR) group. Limited studies have explored the clinical and genomic characteristics of γδ T-ALL, prompting us to conduct a comprehensive analysis of this entity and to identify determinants of outcome. Methods Through a consortium of 13 groups, we assembled a cohort of 200 patients up to 25 years of age with γδ T-ALL enrolled in clinical trials between 2000 and 2018. Clinical data of patients with non-γδ T-ALL enrolled on the same clinical trials were collected (n = 1,067). Complete remission (CR) was defined when bone marrow (BM) showed M1 cytomorphology and/or MRD <1% without evidence of extramedullary disease at end of induction/consolidation (EOI/EOC) and failure to achieve CR was considered treatment failure. A total of 76 γδ T-ALL samples were analyzed by whole genome (WGS) and/or transcriptome (RNAseq) sequencing. Results The frequency of γδ T-ALL was 8.0% of T-ALL cases. Patients with γδ T-ALL exhibited a higher rate of poor prednisone response ( P<0.01), MRD >1% at day 15 ( P<0.01), at EOI ( P<0.01) and EOC ( P<0.01), compared to non-γδ T-ALL cases. Furthermore, patients with γδ T-ALL showed significantly worse 5-year event free survival (EFS, 65% v. 78%, P<0.01) and overall survival (OS, 77% vs 83%, P=0.048). Almost all relapses of γδ T-ALL were isolated BM, while the central nervous system was the main site of relapse in non-γδ T-ALL, suggesting slow treatment response and chemo-resistance to the current treatment in γδ T-ALL. However, γδ T-ALL showed a higher rate of toxic death during treatment (7.6% vs 4.0%, P<0.01), suggesting the need for different therapeutic strategies and risk-classification, rather than treatment intensification. Strikingly, patients less than 3 years of age with γδ T-ALL exhibited significantly poor EFS (33% v. 70% 3-10 years and 73% >10, P<0.01) and OS (49% v. 78% 3-10 and 82% >10, P< 0.01) ( Fig. A), a difference not observed in non-γδ T-ALL. MRD >1% at EOI showed poor EFS (51% v. 96% MRD<0.01% and 91% 1%>MRD>0.01%, P<0.01) and OS (66%). Integrated analysis of WGS and RNAseq identified enrichment of several genomic subtypes in γδ T-ALL, including STAG2/LMO2, hyperdiploidy with recurrent gains of chromosomes 8, 10, 11, 13q and 19, a recently identified “LMO2 γδ-like” subtype with distinct gene expression and LMO2/MYC/MYCN alterations, TLX3-rearranged (-R), and PICALM::MLLT10. No TAL1 nor TLX1-R were detected. STAG2/LMO2 was associated with age at diagnosis before 3 years, and extremely poor outcome, with 4 out of 5 cases dying within three years of diagnosis ( Fig. B). Of 24 STAG2/LMO2 T-ALL (additional 5 non-γδ, 13 TCR unknown cases), 22 of which were diagnosed by age three. All STAG2/LMO2 cases had alterations resulting in LMO2 activation and STAG2 inactivation, most commonly a single rearrangement between these two genes, and upregulation of HBE1, the LIN28-let7 pathway and stem cell proliferation pathways, suggesting a fetal hematopoietic origin. STAG2 has a critical role in the maintenance of enhancer-promoter looping mediated by the cohesin complex. To examine the consequences of STAG2 alterations, we performed integrated genomic/epigenomic analysis of the STAG2/LMO2 (MOLT-14 and PER-117) and STAG2 knockout (KO)/addback T-ALL lines. Chromatin loop sizes defined by H3K27ac HiChIP was highest in STAG2/LMO2 lines compared to other T-ALL. Following restoration of STAG2 expression in MOLT-14, CD34 and ID1/2 were down-regulated and H3K27ac was enriched in pathways related to T-cell differentiation. STAG2 KO in the non- STAG2/LMO2, LMO2-activated line PF382 identified genes also upregulated in STAG2/LMO2 primary samples, including CDK4 and STAG1. STAG2 KO lines exhibited partial compensation of STAG2 binding sites by STAG1 and upregulation of γδ-related genes, RORC and ID1/3. High throughput screening of 2,050 small molecules identified efficacy of HDAC, CDK and PARP inhibitors in STAG2/LMO2 lines. Conclusion Very young onset γδ T-ALL, but not non-γδ T-ALL, is enriched for the STAG2/LMO2 subtype and is a very high risk form of T-ALL. STAG2 loss perturbs chromatin organization and hematopoietic differentiation. Moreover, we demonstrate efficacy of novel therapeutic approaches that are needed to cure this form of leukemia.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background. Philadelphia-Chromosome positive acute lymphoblastic leukemia (Ph+ ALL) is an aggressive disease with a 5-year survival rate of less than 50%. BCR-ABL1 tyrosine kinase inhibitors (TKI) in ...combination with chemotherapy typically fail to induce long-lasting remission in part due to BCR-ABL1 dependent TKI resistance and in part because of BCR-ABL1 independent mechanisms. These findings suggest that the identification of additional targets is necessary for a more effective management of the disease.
Results. We showed previously that Ph+ ALL cells and other types of leukemia are particularly reliant on MYB expression for their growth and survival. To support the essential role of MYB in Ph+ ALL we assessed the biological effects induced by MYB silencing in Ph+ ALL cell lines (BV173 and SUP-B15). In our studies we found that: i) MYB silenced Ph+ ALL cells display reduced growth and colony formation in methylcellulose due to a block in the G1-phase of the cell cycle; ii) such an effect was followed by increased apoptosis detected by Annexin-V staining and caspase-3 cleavage. Moreover, doxycycline-induced MYB silencing in NOD/SCID-IL-2Rγnull (NSG) mice injected with Ph+ ALL cells markedly suppressed leukemia development. These results suggest that MYB would be an ideal candidate for targeted therapies; however, no direct inhibitor for MYB is currently available and little is known about MYB function in primary Ph+ ALL cells. By performing transcriptome analysis of MYB silenced Ph+ ALL cell lines we identified several cell cycle regulatory genes as transcriptional targets of MYB including CDK6, CCND3 and CDKN1A . In particular, CDK6 is critically important for MYB dependent regulation of cell cycle progression because CDK6 silencing induces the cell cycle arrest of Ph+ ALL cells. By contrast, expression of CDK4 was not regulated by MYB and was not required for the proliferation of Ph+ ALL cells, a finding likely explained by the fact that by immunofluorescence, CDK6 is readily detected in the nucleus of Ph+ ALL cells, whereas CDK4 appears exclusively in the cytoplasm. MYB silencing led to a marked decrease in BCL2 expression, concomitantly with the induction of apoptosis. Ectopic expression of BCL2 and CDK6 rescued the growth and survival of Ph+ ALL cells in vitro ; however, it restored in vivo leukemia formation only in part, suggesting that expression of additional MYB targets is required to re-establish the full leukemogenic potential of MYB-silenced cells. We observed that Ph+ ALL cells are remarkably sensitive to CDK4/CDK6 inhibition by Palbociclib confirming the important role of CDK6. On the other hand, the selective BCL2 inhibitor Venetoclax displayed sample-to-sample variation in inducing apoptosis of Ph+ ALL cell lines and primary samples, probably due to variable expression of additional members of the BCL2 family. To overcome this limitation, we examined the anti-leukemia effects of the pan-BCL2 inhibitor Sabutoclax in combination with Palbociclib. The Palbociclib-Sabutoclax combined treatment synergistically reduced the viability of BV173 cells. In addition, NSG mice injected with blast cells from two Ph+ ALL patients were treated for ten days with Palbociclib 150 mg/kg and/or Sabutoclax 5 mg/kg (every other day) and the leukemia burden was analyzed in the peripheral blood before and at bi-weekly intervals after the treatment. By comparing the percentage of circulating leukemic cells before and after the treatment, Palbociclib suppressed Ph+ ALL leukemia development and caused a moderate reduction in the leukemia burden (17% and 32% decrease) while Sabutoclax had negligible effects. However, the Palbociclib-Sabutoclax combination induced a much stronger decrease (77% and 90%), significantly superior to that induced by each drug alone.
Conclusions. These results indicate that the oncogenic effects of MYB in Ph+ ALL is primarily due to the transcriptional activation of CDK6 and BCL2. Furthermore, our xenograft studies constitute a proof-of-principle that the pharmacological targeting of CDK6 and BCL2 is an effective strategy to exploit the MYB “addiction” of Ph+ ALL cells.
Rambaldi:Novartis, Roche/Genentech, Amgen, Italfarmaco: Consultancy; Novartis, Amgen, Celgene, Sanofi: Other: Travel, Accomodations, Expenses. Martinelli:Roche: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Pfizer: Consultancy; Ariad/Incyte: Consultancy; Johnson&Johnson: Consultancy.
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Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold ...targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (
,
,
,
,
and
) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer
internal tandem duplications and phasing double
mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.
The study of minimal residual disease (MRD) in adult patients with acute lymphoblastic leukemia (ALL) allows a greater refinement of the individual risk classification and is the best support for ...risk-specific therapy with or without allogeneic hematopoietic cell transplantation (HCT). Using case-specific sensitive molecular probes or multiparametric flow cytometry on marrow samples obtained from the end of induction until midconsolidation, MRD assays can detect up to 1 leukemic cell of 10,000 total mononuclear cells (sensitivity, 0.01%; ie, ≥104). This cutoff, presently bound to technical limitations and subject to improvement, reflects the individual chemosensitivity and is strongly correlated with treatment outcome. The chance for cure is approximately 70% in the MRD-negative subset but only 20% to 30% in MRD-positive patients, in any diagnostic and risk subset. As shown by prospective trials from Germany, Italy, Spain, and France-Switzerland-Belgium, approximately 50% to 70% of unselected adult patients with Philadelphia-negative ALL achieve and maintain an early MRD response, whereas the remainder do not, including a substantial proportion of clinically standard-risk patients, and require an HCT to avert at least partially the risk of relapse. Along with the diffusion of more effective “pediatric-inspired” chemotherapy programs, the MRD analysis is an integral part of a modern management strategy, guiding the decision process to transplant or not, in which case nonrelapse mortality using HCT in first remission—still 10% to 20%—is totally abolished. The use of new agents such as monoclonal antibodies, small inhibitors, and chimeric antigen receptor T cells is opening a new era of MRD-directed therapies, that will further increase survival rates.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Introduction Patients with secondary acute myeloid leukemia (sAML) after myelodysplastic (MDS) or myeloproliferative neoplasms (MPN) treated with chemotherapy show poorer outcomes compared with de ...novo AML; consequently, these cases should be allocated to allogeneic stem cell transplant (alloSCT) whenever possible (Döhner H, Blood 2017). Some recent evidence suggested the potential of molecular characterization for implementing the current WHO definition (Arber DA, Blood 2016), since chromatin-splicing mutations have been reported to be highly specific for sAML (Lindsley RC, Blood 2015). However, this molecular signature has also been recognized in some clinically defined de novo AML cases (Papaemmanuil E, NEJM 2016). Based on this background, we assessed the clinical impact of chromatin-splicing mutational signature in clinically defined de novo AML patients enrolled into the prospective NILG 02/06 trial ClinicalTrials.gov Identifier: NCT00495287.
Patients and Methods The trial (Bassan R, Blood Advances 2019) randomized 574 newly diagnosed AML patients to receive induction (standard vs high-dose) followed by consolidative chemotherapy and/or alloSCT. For the present analysis, only patients with de novo AML (n=313) and WHO-defined sAML after MDS or MPN (n=101) with a full genetic characterization have been considered. Studies performed at diagnosis included conventional karyotype (n=412) and molecular analysis (n=414) and/or targeted NGS (this latter performed on 197 patients with normal karyotype). Patients with WHO-sAML were defined by the presence of an antecedent history of MDS or MPN (n=21) and/or cytogenetic WHO criteria of AML with MDS-related changes (n=80). Chromatin-splicing mutational signature defined the molecular-sAML group and comprised ASXL1, STAG2, BCOR, KMT2A-PTD, EZH2, PHF6, SRSF2, SF3B1, U2AF1, ZRS2 and RUNX1, excluding patients with WHO recurrent abnormalities.
Results Chromatin-splicing mutations were scored in 55/313 (17.6%) de novo AML patients (hereafter named molecular-sAML). The most frequently reported were KMT2A-PTD (45.5%), RUNX1 (44.4%) and ASXL1 (22.2%), while other mutations in the signature accounted for 5-17.5% of cases. Compared to de novo AML without chromatin-splicing mutations, patients with molecular-sAML and WHO-sAML were older (P<0.0001) and presented with lower white blood cell counts (WBC) (P<0.0001). The 3 groups were balanced in regards to induction regimen (P=0.5) and proportion of patients allocated to a consolidative alloSCT (31% in de novo AML, 30% in WHO-sAML and 33% in molecular-sAML, P=0.8). Complete remission (CR) after 1 or 2 induction cycles was achieved in 93% of de novo AML, 85% of molecular-sAML and 58% of WHO-sAML. In terms of 5-years overall survival (OS) and disease free survival (DFS), de novo AML patients did markedly better than both molecular and WHO-sAML patients (OS: 61%, P<0.0001; DFS: 54%, P<0.0009). Considering sAML patients, WHO-sAML had the worst OS when compared with molecular-sAML (18% vs 30%, P=0.02), but an overlapping DFS (22% vs 26%, P<0.3) (Figure 1). The negative impact of chromatin-splicing mutations was independently confirmed by multivariate analysis accounting for age, performance status, WBC and induction regimen HR 2.2 (CI 95% 1.48-3.25), P=0.0001. Among chromatin-splicing mutations, only RUNX1 and U2AF1 significantly affected OS HR 3.55 (CI 95% 1.28-9.87), P=0.01 and HR 6.87 (CI 95% 1.71-27.55), P=0.006. Finally, a consolidative alloSCT improved survival in all patients groups, most significantly in molecular and WHO-sAML (48% vs 24%, P=0.07 and 38% vs 8%, P=0.0001, respectively).
Conclusions Chromatin-splicing mutational signature identifies a distinct high-risk group within de novo AML patients, which shows clinical characteristics and outcomes closer to sAML than to de novo AML patients. These data highlight the need to detect this molecular signature at diagnosis and support a molecular definition of sAML.
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Ferrero:Novartis: Honoraria. Corradini:Celgene: Honoraria, Other: Travel Costs; Gilead: Honoraria, Other: Travel Costs; Jazz Pharmaceutics: Honoraria; KiowaKirin: Honoraria; Kite: Honoraria; Novartis: Honoraria, Other: Travel Costs; Daiichi Sankyo: Honoraria; AbbVie: Consultancy, Honoraria, Other: Travel Costs; Amgen: Honoraria; Janssen: Honoraria, Other: Travel Costs; Roche: Honoraria; Sanofi: Honoraria; Servier: Honoraria; Takeda: Honoraria, Other: Travel Costs; BMS: Other: Travel Costs. Bassan:Incyte: Honoraria; Amgen Inc.: Honoraria; Pfizer: Honoraria; Shire: Honoraria. Rambaldi:Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support.
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