Therapy-Related Myeloid Neoplasm: Biology and Mechanistic Aspects of Malignant Progression.

Therapy-related myeloid neoplasms (t-MN) arise after a documented history of chemo/radiotherapy as treatment for an unrelated condition and account for 10-20% of myelodysplastic syndromes and acute myeloid leukemia. T-MN are characterized by a specific genetic signature, aggressive features and dismal prognosis. The nomenclature and the subsets of these conditions have changed frequently over time, and despite the fact that, in the last classification, they lost their autonomous entity status and became disease qualifiers, the recognition of this feature remains of major importance. Furthermore, in recent years, extensive studies focusing on clonal hematopoiesis and germline variants shed light on the mechanisms of positive pressure underpinning the rise of driver gene mutations in t-MN. In this manuscript, we aim to review the evolution of defining criteria and characteristics of t-MN from a clinical and biological perspective, the advances in mechanistic aspects of malignant progression and the challenges in prevention and management.

Retinoic acid and proteotoxic stress induce AML cell death overcoming stromal cell protection.

BACKGROUND: Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A). METHODS: We treated FLT3-ITD+ AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD+ AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML. RESULTS: The combination RBA exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs. CONCLUSIONS: Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.

YY1 Knockdown Relieves the Differentiation Block and Restores Apoptosis in AML Cells.

In this study we analyzed the expression of Yin and Yang 1 protein (YY1), a member of the noncanonical PcG complexes, in AML patient samples and AML cell lines and the effect of YY1 downregulation on the AML differentiation block. Our results show that YY1 is significantly overexpressed in AML patient samples and AML cell lines and that YY1 knockdown relieves the differentiation block. YY1 downregulation in two AML cell lines (HL-60 and OCI-AML3) and one AML patient sample restored the expression of members of the CEBP protein family, increased the expression of extrinsic growth factors/receptors and surface antigenic markers, induced morphological cell characteristics typical of myeloid differentiation, and sensitized cells to retinoic acid treatment and to apoptosis. Overall, our data show that YY1 is not a secondary regulator of myeloid differentiation but that, if overexpressed, it can play a predominant role in myeloid differentiation block.

Applicability of 2022 classifications of acute myeloid leukemia in the real-world setting.

The increasing knowledge of molecular genetics of acute myeloid leukemia (AML) necessitated the update of previous diagnostic and prognostic schemes, which resulted in the development of the World Health Organization (WHO), the International Consensus Classification (ICC), and the new European LeukemiaNet (ELN) recommendations in 2022. We aimed to provide a real-world application of the new models, unravel differences and similarities, and test their implementation in clinical AML diagnosis. A total of 1001 patients diagnosed with AML were reclassified based on the new schemes. The overall diagnostic changes between the WHO 2016 and the WHO 2022 and ICC classifications were 22.8% and 23.7%, respectively, with a 13.1% difference in patients' distribution between ICC and WHO 2022. The 2022 ICC "not otherwise specified" and WHO "defined by differentiation" AML category sizes shrank when compared with that in WHO 2016 (24.1% and 26.8% respectively, vs 38.7%), particularly because of an expansion of the myelodysplasia (MDS)-related group. Of 397 patients with a MDS-related AML according to the ICC, 55.9% were defined by the presence of a MDS-related karyotype. The overall restratification between ELN 2017 and ELN 2022 was 12.9%. The 2022 AML classifications led to a significant improvement of diagnostic schemes. In the real-world setting, conventional cytogenetics, usually rapidly available and less expensive than molecular characterization, stratified 56% of secondary AML, still maintaining a powerful diagnostic role. Considering the similarities between WHO and ICC diagnostic schemes, a tentative scheme to generate a unified model is desirable.

MCL1 regulates AML cells metabolism via direct interaction with HK2. Metabolic signature at onset predicts overall survival in AMLs' patients.

We characterize the metabolic background in distinct Acute Myeloid Leukemias (AMLs), by comparing the metabolism of primary AML blasts isolated at diagnosis with that of normal hematopoietic maturing progenitors, using the Seahorse XF Agilent. Leukemic cells feature lower spare respiratory (SRC) and glycolytic capacities as compared to hematopoietic precursors (i.e. day 7, promyelocytes). According with Proton Leak (PL) values, AML blasts can be grouped in two well defined populations. The AML group with blasts presenting high PL or high basal OXPHOS plus high SRC levels had shorter overall survival time and significantly overexpressed myeloid cell leukemia 1 (MCL1) protein. We demonstrate that MCL1 directly binds to Hexokinase 2 (HK2) on the outer mitochondrial membrane (OMM). Overall, these results suggest that high PL and high SRC plus high basal OXPHOS levels at disease onset, arguably with the concourse of MCL1/HK2 action, are significantly linked with shorter overall survival time in AML. Our data describe a new function for MCL1 protein in AMLs' cells: by forming a complex with HK2, MCL1 co-localizes to VDAC on the OMM, thus inducing glycolysis and OXPHOS, ultimately conferring metabolic plasticity and promoting resistance to therapy.

Impairment of FOXM1 expression in mesenchymal cells from patients with myeloid neoplasms, de novo and therapy-related, may compromise their ability to support hematopoiesis.

Bone marrow mesenchymal stem cells (BM-MSCs) exhibit multiple abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including reduced proliferative and clonogenic capacity, altered morphology, impaired immunoregulatory properties and capacity to support hematopoiesis. Here, we investigated expression of the FOXM1 gene, a transcription factor driving G2/M gene expression, in BM-MSCs isolated from patients with MDS and AML, de novo and therapy-related, compared to BM-MSCs isolated from healthy donors (HD). We observed a statistically significant downregulation of FOXM1 expression in BM-MSCs isolated from MDS and AML patients, as compared to controls. In parallel, expression of FOXM1 mitotic targets (CCNB1, CDC20, PLK1 and NDC80) was suppressed in patients' BM-MSCs, as compared to HD. No differences in the expression of FOXM1 and its mitotic targets were observed in BM-mononuclear cells from the different sources. From a functional standpoint, silencing of FOXM1 mRNA in healthy MSC induced a significant decrease in the expression of its targets. In this line, healthy MSC silenced for FOXM1 showed an impaired ability to support hematopoiesis in vitro. These findings suggest that deregulation of FOXM1 may be involved in the senescent phenotype observed in MSC derived from myeloid neoplasms.

An Polycythemia Vera Evolve from Acute Myeloid Leukemia? Report of a Case Showing a Simultaneous Minor JAK2 V617F Mutated Clone.

The evolution of myeloproliferative neoplasms (MPN) to acute myeloid leukemia (AML) occurs in 2-10% of patients, depending on the MPN subtype, treatment, and follow-up length. The reverse-path from AML to MPN has been rarely reported. We herein present a 75 years old woman with AML, in whom a JAK2-V617F positive polycythemia vera (PV) emerged during follow-up, 19 months from the end of consolidation treatment. JAK2-V617F mutation screening retrospectively performed by Next Generation Sequencing (NGS) and JAK2 MutaScreen was negative on the bone marrow sample collected at AML diagnosis. However, using digital droplet PCR (ddPCR), we detected a minor JAK2 V617F mutated clone at AML onset. In addition, a TET2 R550 mutated clone persisted at stable levels throughout the disease course. This case shows that a very small MPN clone masked at AML diagnosis may expand after treatment end and be erroneously interpreted as MPN evolving from AML. Very sensitive techniques such as ddPCR may help to unravel the true disease history in these cases.

Vitamin C Deficiency in Patients With Acute Myeloid Leukemia.

Vitamin C has been shown to play a significant role in suppressing progression of leukemia through epigenetic mechanisms. We aimed to study the role of vitamin C in acute myeloid leukemia (AML) biology and clinical course. To this purpose, the plasma levels of vitamin C at diagnosis in 62 patients with AML (including 5 cases with acute promyelocytic leukemia, APL),7 with myelodysplastic syndrome (MDS), and in 15 healthy donors (HDs) were studied. As controls, vitamins A and E levels were analysed. Expression of the main vitamin C transporters and of the TET2 enzyme were investigated by a specific RQ-PCR while cytoplasmic vitamin C concentration and its uptake were studied in mononuclear cells (MNCs), lymphocytes and blast cells purified from AML samples, and MNCs isolated from HDs. There were no significant differences in vitamin A and E serum levels between patients and HDs. Conversely, vitamin C concentration was significantly lower in AML as compared to HDs (p<0.0001), inversely correlated with peripheral blast-counts (p=0.029), significantly increased at the time of complete remission (CR) (p=0.04) and further decreased in resistant disease (p=0.002). Expression of the main vitamin C transporters SLC23A2, SLC2A1 and SLC2A3 was also significantly reduced in AML compared to HDs. In this line, cytoplasmic vitamin C levels were also significantly lower in AML-MNCs versus HDs, and in sorted blasts compared to normal lymphocytes in individual patients. No association was found between vitamin C plasma levels and the mutation profile of AML patients, as well as when considering cytogenetics or 2017 ELN risk stratification groups. Finally, vitamin C levels did not play a predictive role for overall or relapse-free survival. In conclusion, our study shows that vitamin C levels are significantly decreased in patients with AML at the time of initial diagnosis, further decrease during disease progression and return to normal upon achievement of CR. Correspondingly, low intracellular levels may mirror increased vitamin C metabolic consumption in proliferating AML cells.

Ascorbate Plus Buformin in AML: A Metabolic Targeted Treatment.

In the present study, we characterized the metabolic background of different Acute Myeloid Leukemias' (AMLs) cells and described a heterogeneous and highly flexible energetic metabolism. Using the Seahorse XF Agilent, we compared the metabolism of normal hematopoietic progenitors with that of primary AML blasts and five different AML cell lines. We assessed the efficacy and mechanism of action of the association of high doses of ascorbate, a powerful oxidant, with the metabolic inhibitor buformin, which inhibits mitochondrial complex I and completely shuts down mitochondrial contributions in ATP production. Primary blasts from seventeen AML patients, assayed for annexin V and live/dead exclusion by flow cytometry, showed an increase in the apoptotic effect using the drug combination, as compared with ascorbate alone. We show that ascorbate inhibits glycolysis through interfering with HK1/2 and GLUT1 functions in hematopoietic cells. Ascorbate combined with buformin decreases mitochondrial respiration and ATP production and downregulates glycolysis, enhancing the apoptotic effect of ascorbate in primary blasts from AMLs and sparing normal CD34+ bone marrow progenitors. In conclusion, our data have therapeutic implications especially in fragile patients since both agents have an excellent safety profile, and the data also support the clinical evaluation of ascorbate-buformin in association with different mechanism drugs for the treatment of refractory/relapsing AML patients with no other therapeutic options.

Atypical Rearrangements in APL-Like Acute Myeloid Leukemias: Molecular Characterization and Prognosis.

Acute promyelocytic leukemia (APL) accounts for 10-15% of newly diagnosed acute myeloid leukemias (AML) and is typically caused by the fusion of promyelocytic leukemia with retinoic acid receptor α (RARA) gene. The prognosis is excellent, thanks to the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) combination therapy. A small percentage of APLs (around 2%) is caused by atypical transcripts, most of which involve RARA or other members of retinoic acid receptors (RARB or RARG). The diagnosis of these forms is difficult, and clinical management is still a challenge for the physician due to variable response rates to ATRA and ATO. Herein we review variant APL cases reported in literature, including genetic landscape, incidence of coagulopathy and differentiation syndrome, frequent causes of morbidity and mortality in these patients, sensitivity to ATRA, ATO, and chemotherapy, and outcome. We also focus on non-RAR rearrangements, complex rearrangements (involving more than two chromosomes), and NPM1-mutated AML, an entity that can, in some cases, morphologically mimic APL.

From Clonal Hematopoiesis to Therapy-Related Myeloid Neoplasms: The Silent Way of Cancer Progression.

Clonal hematopoiesis (CH) has been recognized as a predisposing factor for the development of myeloid malignancies. Its detection has been reported at different frequencies across studies, based on the type of genome scanning approach used and the population studied, but the latest insights recognize its virtual ubiquitous presence in older individuals. The discovery of CH in recent years paved the way for a shift in the paradigm of our understanding of the biology of therapy-related myeloid malignancies (t-MNs). Indeed, we moved from the concept of a treatment-induced lesion to a model where CH precedes the commencement of any cancer-related treatment in patients who subsequently develop a t-MN. Invariant patterns of genes seem to contribute to the arising of t-MN cases, with differences regarding the type of treatment received. Here, we review the principal studies concerning CH, the relationship with myeloid progression and the mechanisms of secondary t-MN development.

Terminal deoxynucleotidyl transferase (TdT) expression is associated with FLT3-ITD mutations in Acute Myeloid Leukemia.

The terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in acute myeloid leukemias (AMLs), where it may be involved in the generation of NPM1 and FLT3-ITD mutations. We studied the correlations between TdT expression and FLT3-ITD or NPM1 mutations in primary AML samples, and the impact on patients' survival. TdT expression was analyzed in 143 adult AML patients by flow cytometry as percentage of positivity and mean fluorescence intensity (MFI) on blasts. TdT was positive in 49 samples (34.2%), with a median of 48% TdT-positivity (range 7-98) and a median MFI of 2.70 (range 1.23-30.54). FLT3-ITD and NPM1 mutations were present in 24 (16.7%) and 34 (23.7%) cases, respectively. Median TdT expression on blasts was significantly higher in FLT3-ITD+, as compared with FLT3-ITD- AMLs (median 8% vs 0% respectively, p = 0.035). NPM1 mutational status, FLT3-ITD allelic ratio, karyotype, and ELN risk groups, did not correlate with TdT expression or MFI on blasts. TdT + patients had poorer survival as compared to TdT-, but this result was not confirmed by the multivariable analysis, where ELN risk stratification as well as age and type of treatment remained independent prognostic factors for OS. In summary, our results support the possible implication of TdT enzyme in the generation of FLT3-ITD mutations in AML.

Characterization of FLT3-ITDmut acute myeloid leukemia: molecular profiling of leukemic precursor cells.

Acute myeloid leukemia (AML) with FLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3-ITD-positivity. The aim of this study was to characterize the distribution of FLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3-ITDmut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3-ITDmut AML (n = 12). A higher FLT3-ITDmut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/-,CD25-,CD99low/-) (p = 0.0005) and mononuclear cells (MNCs) (p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higher FLT3-ITDmut burden was also observed in LPCs of AML patients with a small FLT3-ITDmut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3-ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3-ITDmut AML.

Retinoic acid synergizes with the unfolded protein response and oxidative stress to induce cell death in FLT3-ITD+ AML.

Acute myeloid leukemia (AML) is often characterized by the expression of fusion or mutant proteins that cause impaired differentiation and enhanced proliferation and survival. The presence of mutant proteins prone to misfolding can render the cells sensitive to endoplasmic reticulum (ER) stress and oxidative stress that could otherwise be overcome. Here, we show that the triple combination of the differentiating agent retinoic acid (RA), the ER stress-inducing drug tunicamycin (Tm), and arsenic trioxide (ATO), able to generate oxidative stress, leads to the death of AML cell lines expressing fusion proteins involving the gene MLL and the internal tandem duplication (ITD) in the FLT3 tyrosine kinase receptor. Importantly, the combination of RA, Tm, and ATO decreased the colony-forming capacity of primary leukemic blasts bearing the FLT-ITD mutation without affecting healthy hematopoietic progenitor cells. We demonstrate in cell lines that combination of these drugs generates ER and oxidative stresses and impairs maturation and causes accumulation of FLT3 protein in the ER. Our data provide a proof of concept that low amounts of drugs that generate ER and oxidative stresses combined with RA could be an effective targeted therapy to hit AML cells characterized by MLL fusion proteins and FLT3-ITD mutation.

Mutational landscape of patients with acute promyelocytic leukemia at diagnosis and relapse.

Despite the high probability of cure of patients with acute promyelocytic leukemia (APL), mechanisms of relapse are still largely unclear. Mutational profiling at diagnosis and/or relapse may help to identify APL patients needing frequent molecular monitoring and early treatment intervention. Using an NGS approach including a 31 myeloid gene-panel, we tested BM samples of 44 APLs at the time of diagnosis, and of 31 at relapse. Mutations in PML and RARA genes were studied using a customized-NGS-RNA panel. Patients relapsing after ATRA-chemotherapy rarely had additional mutations (P = .009). In patients relapsing after ATRA/ATO, the PML gene was a preferential mutation target. We then evaluated the predictive value of mutations at APL diagnosis. A median of two mutations was detectable in 9/11 patients who later relapsed, vs one mutation in 21/33 patients who remained in CCR (P = .0032). This corresponded to a significantly lower risk of relapse in patients with one or less mutations (HR 0.046; 95% CI 0.011-0.197; P < .0001). NGS-analysis at the time of APL diagnosis may inform treatment decisions, including alternative treatments for cases with an unfavorable mutation profile.

PML/RARa Interferes with NRF2 Transcriptional Activity Increasing the Sensitivity to Ascorbate of Acute Promyelocytic Leukemia Cells.

: NRF2 (NF-E2 p45-related factor 2) orchestrates cellular adaptive responses to stress. Its quantity and subcellular location is controlled through a complex network and its activity increases during redox perturbation, inflammation, growth factor stimulation, and energy fluxes. Even before all-trans retinoic acid (ATRA) treatment era it was a common experience that acute promyelocytic leukemia (APL) cells are highly sensitive to first line chemotherapy. Since we demonstrated how high doses of ascorbate (ASC) preferentially kill leukemic blast cells from APL patients, we aimed to define the underlying mechanism and found that promyelocytic leukemia/retinoic acid receptor α (PML/RARa) inhibits NRF2 function, impedes its transfer to the nucleus and enhances its degradation in the cytoplasm. Such loss of NRF2 function alters cell metabolism, demarcating APL tissue from both normal promyelocytes and other acute myeloide leukemia (AML) blast cells. Resistance to ATRA/arsenic trioxide (ATO) treatment is rare but grave and the metabolically-oriented treatment with high doses of ASC, which is highly effective on APL cells and harmless on normal hematopoietic stem cells (HSCs), could be of use in preventing clonal evolution and in rescuing APL-resistant patients.

Identification and monitoring of atypical PML/RARA fusion transcripts in acute promyelocytic leukemia.

Once the diagnostic suspicion of acute promyelocytic leukemia (APL) has been raised, international guidelines recommend prompt initiation of tailored therapy and supportive care, while awaiting for genetic confirmation of the diagnosis, and the identification of the specific PML/RARA isoform by reverse transcriptase polymerase chain reaction (RT-PCR). Depending on the PML break point, usually located within intron 6, exon 6, or intron 3, different PML/RARA transcript isoforms may be generated, that is, long (bcr1), variant (bcr2), and short (bcr3), respectively. We report here the characterization of three APL cases harboring atypical PML/RARA transcripts, which were not clearly detectable after standard RT-PCR amplification. In all three cases, clinical, morphological, and immunophenotypic features were consistent with APL. Direct sequencing allowed the identification of atypical break points within the PML and RARA genes. Then, we designed a patient-specific quantitative real-time PCR for the atypical transcripts, which allowed for specific quantitative evaluation of minimal residual disease (MRD) during follow-up. Despite the rarity of APL cases with an atypical PML/RARA fusion, our study indicates that an integrated laboratory approach, employing several diagnostic techniques is crucial to timely diagnose APL. This approach allows prompt initiation of specific targeted treatment and reliable MRD monitoring in atypical APL cases.