Deferasirox-Dependent Iron Chelation Enhances Mitochondrial Dysfunction and Restores p53 Signaling by Stabilization of p53 Family Members in Leukemic Cells.

Iron is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.

Illuminating novel biological aspects and potential new therapeutic approaches for chronic myeloproliferative malignancies.

This review reflects the presentations and discussion at the 14th post-American Society of Hematology (ASH) International Workshop on Chronic Myeloproliferative Malignancies, which took place on the December 10 and 11, 2019, immediately after the 61st ASH Annual Meeting in Orlando, Florida. Rather than present a resume of the proceedings, we address some of the topical translational science research and clinically relevant topics in detail. We consider how recent studies using single-cell genomics and other molecular methods reveal novel aspects of hematopoiesis which in turn raise the possibility of new therapeutic approaches for patients with myeloproliferative neoplasms (MPNs). We discuss how alternative therapies could benefit patients with chronic myeloid leukemia who develop BCR-ABL1 mutant subclones following ABL1-tyrosine kinase inhibitor therapy. In MPNs, we focus on efforts beyond JAK-STAT and the merits of integrating activin receptor ligand traps, interferon-α, and allografting in the current treatment algorithm for patients with myelofibrosis.

Mimicking the Acute Myeloid Leukemia Niche for Molecular Study and Drug Screening.

Bone marrow niche is a major contributing factor in leukemia development and drug resistance in acute myeloid leukemia (AML) patients. Although mimicking leukemic bone marrow niche relies on two-dimensional (2D) culture conditions, it cannot recapitulate complex bone marrow structure that causes introduction of different three-dimensional (3D) scaffolds. Simultaneously, microfluidic platform by perfusing medium culture mimic interstitial fluid flow, along with 3D scaffold would help for mimicking bone marrow microenvironment. In this study TF-1 cells were cocultured with bone marrow mesenchymal stem cells (BM-MSCs) in 2D and 3D microfluidic devices. Phenotype maintenance during cell culture and proliferation rate was assayed and confirmed by cell cycle analysis. Morphology of cells in 2D and 3D culture conditions was demonstrated by scanning electron microscopy. After these experiments, drug screening was performed by applying azacitidine and cytarabine and cytotoxicity assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for B cell lymphoma 2 (BCL2) were done to compare drug resistance in 2D and 3D culture conditions. Our result shows leukemic cells in 3D microfluidic device retaining their phenotype and proliferation rate was significantly higher in 3D culture condition in comparison to 2D culture condition (p < 0.05), which was confirmed by cell cycle analysis. Cytotoxicity assay also illustrated drug resistance in 3D culture condition and qRT-PCR demonstrated higher BCL2 expression in 3D microfluidic device in contrast to 2D microfluidic device (p < 0.05). On balance, mimicking bone marrow niche would help the target therapy and specify the role of niche in development of leukemia in AML patients.

Milestones and monitoring.

In chronic myeloid leukemia (CML), the presence of a specific chromosome marker (Ph-chromosome) as well as of the corresponding molecular marker (BCR-ABL fusion transcripts) provides suitable and precise tools to monitor the burden of the disease present at diagnosis and that of the residual disease present at specific time points during treatment. A huge number of studies have clearly demonstrated that in CML cytogenetic and molecular responses are strictly correlated to the final outcome of the patients and the correct use of standardized methods to assess the achievement of specific degrees of disease reduction at specific time points during treatment has become an essential part of proper clinical management of CML. The target to be achieved and the corresponding "optimal response" definition are however evolving, and at least for some patients, they may be represented not only by best possible overall survival (OS) but also by the possibility to discontinue the tyrosine-kinase inhibitor (TKI) treatment and therefore to live in a treatment-free remission (TFR) status. Therefore, at least for some patients, deep degrees of molecular response, as MR(4) and MR(4.5), whose precise definition has been recently introduced and that are prerequisites to try to discontinuation, are becoming the target to be achieved even in common clinical practice. As a fast initial decline of the disease burden after therapy start may be highly predictive for the final outcome of patients not only in terms of progression-free survival (PFS) and of PS but also in terms of possibility of achieving deep molecular responses, a more intense and punctual monitoring of the response of CML patients during the first 6 months of TKI therapy is now recommended by the more recent versions of the European Leukemia Net (ELN) and National Comprehensive Cancer Network (NCCN) guidelines, as this represents the major driver to decide therapy.

Selective strong synergism of Ruxolitinib and second generation tyrosine kinase inhibitors to overcome bone marrow stroma related drug resistance in chronic myelogenous leukemia.

The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co-cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.

Initial molecular response at 3 months may predict both response and event-free survival at 24 months in imatinib-resistant or -intolerant patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase treated with nilotinib.

PURPOSE: The association between initial molecular response and longer-term outcomes with nilotinib was examined. PATIENTS AND METHODS: Patients with imatinib-resistant or -intolerant chronic myeloid leukemia in chronic phase from the phase II nilotinib registration study with available postbaseline BCR-ABL1 transcript assessments were included (N = 237). RESULTS: BCR-ABL1 transcript levels (International Scale [IS]) at 3 months correlated with complete cytogenetic response (CCyR) by 24 months. Patients with BCR-ABL1 (IS) of > 1% to ≤ 10% at 3 months with nilotinib had higher cumulative incidence of CCyR by 24 months than patients with BCR-ABL1 (IS) of > 10% (53% v 16%). BCR-ABL1 (IS) at 3 months predicted major molecular response (MMR) by 24 months. Cumulative incidence of MMR by 24 months for patients with BCR-ABL1 (IS) of > 0.1% to ≤ 1%, > 1% to ≤ 10%, and > 10% was 65%, 27%, and 9%, respectively. These differences were observed for patients with or without baseline BCR-ABL1 mutations and for those with imatinib resistance or intolerance. Estimated event-free survival (EFS) rates at 24 months decreased with higher transcript levels at 3 months; patients with BCR-ABL1 (IS) of ≤ 1% had an estimated 24-month EFS rate of 82%, compared with 70% for patients with BCR-ABL1 (IS) of > 1% to ≤ 10% and 48% for patients with BCR-ABL1 (IS) of > 10%. CONCLUSION: Patients with BCR-ABL1 (IS) of > 10% at 3 months had a lower cumulative incidence of CCyR and MMR and lower rates of EFS versus patients with BCR-ABL1 (IS) of ≤ 10%. Prospective studies may determine whether close monitoring or alternative therapies are warranted for patients with minimal initial molecular response.

Iron chelation therapy in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous disorder of the hematopoietic stem cells, frequently characterized by anemia and transfusion dependency. In low-risk patients, transfusion dependency can be long lasting, leading to iron overload. Iron chelation therapy may be a therapeutic option for these patients, especially since the approval of oral iron chelators, which are easier to use and better accepted by the patients. The usefulness of iron chelation in MDS patients is still under debate, mainly because of the lack of solid prospective clinical trials that should take place in the future. This review aims to summarize what is currently known about the incidence and clinical consequences of iron overload in MDS patients and the state-of the-art of iron chelation therapy in this setting. We also give an overview of clinical guidelines for chelation in MDS published to date and some perspectives for the future.

Deferasirox is a powerful NF-kappaB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging.

BACKGROUND: Usefulness of iron chelation therapy in myelodysplastic patients is still under debate but many authors suggest its possible role in improving survival of low-risk myelodysplastic patients. Several reports have described an unexpected effect of iron chelators, such as an improvement in hemoglobin levels, in patients affected by myelodysplastic syndromes. Furthermore, the novel chelator deferasirox induces a similar improvement more rapidly. Nuclear factor-kappaB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including myelodysplastic syndromes. DESIGN AND METHODS: We evaluated deferasirox activity on nuclear factor-kappaB in myelodysplastic syndromes as a possible mechanism involved in hemoglobin improvement during in vivo treatment. Forty peripheral blood samples collected from myelodysplastic syndrome patients were incubated with 50 muM deferasirox for 18h. RESULTS: Nuclear factor-kappaB activity dramatically decreased in samples showing high basal activity as well as in cell lines, whereas no similar behavior was observed with other iron chelators despite a similar reduction in reactive oxygen species levels. Additionally, ferric hydroxyquinoline incubation did not decrease deferasirox activity in K562 cells suggesting the mechanism of action of the drug is independent from cell iron deprivation by chelation. Finally, incubation with both etoposide and deferasirox induced an increase in K562 apoptotic rate. CONCLUSIONS: Nuclear factor-kappaB inhibition by deferasirox is not seen from other chelators and is iron and reactive oxygen species scavenging independent. This could explain the hemoglobin improvement after in vivo treatment, such that our hypothesis needs to be validated in further prospective studies.

Identification of different Ikaros cDNA transcripts in Philadelphia-positive adult acute lymphoblastic leukemia by a high-throughput capillary electrophoresis sizing method.

BACKGROUND: Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA. DESIGN AND METHODS: In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts. RESULTS: We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus. CONCLUSIONS: Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia.