Microenvironment in acute myeloid leukemia: focus on senescence mechanisms, therapeutic interactions, and future directions.

Acute myeloid leukemia (AML) is a disease with a dismal prognosis, mainly affecting the elderly. In recent years, new drugs have improved life expectancy and quality of life, and a better understanding of the genetic-molecular nature of the disease has shed light on previously unknown aspects of leukemogenesis. In parallel, increasing attention has been attracted to the complex interactions between cells and soluble factors in the bone marrow (BM) environment, collectively known as the microenvironment. In this review, we discuss the central role of the microenvironment in physiologic and pathologic hematopoiesis and the mechanisms of senescence, considered a fundamental protective mechanism against the proliferation of damaged and pretumoral cells. The microenvironment also represents a fertile ground for the development of myeloid malignancies, and the leukemic niche significantly interacts with drugs commonly used in AML treatment. Finally, we focus on the role of the microenvironment in the engraftment and complications of allogeneic hematopoietic stem cell transplantation, the only curative option in a conspicuous proportion of patients.

Diagnostic capabilities, clinical features, and longitudinal UBA1 clonal dynamics of a nationwide VEXAS cohort.

VEXAS is a prototypic hemato-inflammatory disease combining rheumatologic and hematologic disorders in a molecularly defined nosological entity. In this nationwide study, we aimed at screenshotting the current diagnostic capabilities and clinical-genomic features of VEXAS, and tracked UBA1 longitudinal clonal dynamics upon different therapeutics, including allogeneic hematopoietic cell transplant. We leveraged a collaboration between the Italian Society of Experimental Hematology and of Rheumatology and disseminated a national survey to collect clinical and molecular patient information. Overall, 13/29 centers performed UBA1 genomic testing locally, including Sanger sequencing (46%), next-generation sequencing (23%), droplet digital polymerase chain reaction (8%), or combination (23%). A total of 41 male patients were identified, majority (51%) with threonine substitutions at Met41 hotspot, followed by valine and leucine (27% and 8%). Median age at VEXAS diagnosis was 67 years. All patients displayed anemia (median hemoglobin 9.1 g/dL), with macrocytosis. Bone marrow vacuoles were observed in most cases (89%). The most common rheumatologic association was polychondritis (49%). A concomitant myelodysplastic neoplasm/syndrome (MDS) was diagnosed in 71% of patients (n = 28), chiefly exhibiting lower Revised International Prognostic Scoring System risk profiles. Karyotype was normal in all patients, except three MDS cases showing -Y, t(12;16)(q13;q24), and +8. The most frequently mutated gene was DNMT3A (n = 10), followed by TET2 (n = 3). At last follow-up, five patients died and two patients progressed to acute leukemia. Longitudinal UBA1 clonal dynamics demonstrated mutational clearance following transplant. We collected a nationwide interdisciplinary VEXAS patient cohort, characterized by heterogeneous rheumatologic manifestations and treatments used. MDS was diagnosed in 71% of cases. Patients exhibited various longitudinal UBA1 clonal dynamics.

Bone Marrow Microenvironment Involvement in t-MN: Focus on Mesenchymal Stem Cells.

Therapy-related myeloid neoplasms (t-MN) are a late complication of cytotoxic therapy (CT) used in the treatment of both malignant and non-malignant diseases. Historically, t-MN has been considered to be a direct consequence of DNA damage induced in normal hematopoietic stem or progenitor cells (HSPC) by CT. However, we now know that treatment-induced mutations in HSC are not the only players involved in t-MN development, but additional factors may contribute to the onset of t-MN. One of the known drivers involved in this field is the bone marrow microenvironment (BMM) and, in particular, bone marrow mesenchymal stem cells (BM-MSC), whose role in t-MN pathogenesis is the topic of this mini-review. BM-MSCs, physiologically, support HSC maintenance, self-renewal, and differentiation through hematopoietic-stromal interactions and the production of cytokines. In addition, BM-MSCs maintain the stability of the BM immune microenvironment and reduce the damage caused to HSC by stress stimuli. In the t-MN context, chemo/radiotherapy may induce damage to the BM-MSC and likewise alter BM-MSC functions by promoting pro-inflammatory response, clonal selection and/or the production of factors that may favor malignant hematopoiesis. Over the last decade, it has been shown that BM-MSC isolated from patients with de novo and therapy-related MN exhibit decreased proliferative and clonogenic capacity, altered morphology, increased senescence, defective osteogenic differentiation potential, impaired immune-regulatory properties, and reduced ability to support HSC growth and differentiation, as compared to normal BM-MSC. Although the understanding of the genetic and gene expression profile associated with ex vivo-expanded t-MN-MSCs remains limited and debatable, its potential role in prognostic and therapeutic terms is acting as a flywheel of attraction for many researchers.

Correlation analysis between auto-immunological and mutational profiles in myelodysplastic syndromes.

OBJECTIVE AND DESIGN: Systemic-Inflammatory-Autoimmune-Diseases (SIAD) is increasingly considered in Myelodysplastic-Syndromes (MDS). In this line, we evaluated the MDS auto-immunological profile, correlating it to the mutational landscape, trying to identify a molecular-genetic trigger agent related to SIAD. METHODS AND MATERIALS: Eighty-one MDS were enrolled and t-NGS was performed. Anti-Nuclear-Antibodies (ANA) were tested, and ANA-antigenic-specificity was characterized by ANA-profile, ENA-screen, anti-dsDNA. Non-Hematological-Patients (NHP) and Healthy-Donors (HD) were used as controls. RESULTS: At clinically relevant cut-off (≥ 1:160), ANA was significantly more frequent in MDS, while ANA-antigenic-specificity showed a low association rate. ANA ≥ 1:160-positive MDS showed a mutational landscape similar to ANA-negative/ANA < 1:160 MDS. No significant correlations between mutational and immunological profiles were found and UBA1 mutations, related to VEXAS, were absent. CONCLUSIONS: Although ANA-positivity was found to be increased in MDS, the low ANA-antigenic-specificity suggests that autoantibodies didn't recognize autoimmune-pathognomonic antigens. The lack of relationship between genetic profile and ANA-positivity, suggests that MDS genetic variants may not be the direct cause of SIAD.

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.

Demethylation and Up-Regulation of an Oncogene after Hypomethylating Therapy.

BACKGROUND: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. METHODS: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. RESULTS: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. CONCLUSIONS: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

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.

Clonal haematopoiesis as a risk factor for therapy-related myeloid neoplasms in patients with chronic lymphocytic leukaemia treated with chemo-(immuno)therapy.

Clonal haematopoiesis of indeterminate potential (CHIP) may predispose for the development of therapy-related myeloid neoplasms (t-MN). Using target next-generation sequencing (t-NGS) panels and digital droplet polymerase chain reactions (ddPCR), we studied the myeloid gene mutation profiles of patients with chronic lymphocytic leukaemia (CLL) who developed a t-MN after treatment with chemo-(immuno)therapy. Using NGS, we detected a total of 30 pathogenic/likely pathogenic (P/LP) variants in 10 of 13 patients with a t-MN (77%, median number of variants for patient: 2, range 0-6). The prevalence of CHIP was then backtracked in paired samples taken at CLL diagnosis in eight of these patients. Six of them carried at least one CHIP-variant at the time of t-MN (median: 2, range: 1-5), and the same variants were present in the CLL sample in five cases. CHIP variants were present in 34 of 285 patients from a population-based CLL cohort, which translates into a significantly higher prevalence of CHIP in patients with a CLL who developed a t-MN, compared to the population-based cohort (5/8, 62.5% vs. 34/285, 12%, p = 0.0001). Our data show that CHIP may be considered as a novel parameter affecting treatment algorithms in patients with CLL, and highlight the potential of using chemo-free therapies in CHIP-positive cases.

Mutational profile of ZBTB16-RARA-positive acute myeloid leukemia.

BACKGROUND: The ZBTB16-RARA fusion gene, resulting from the reciprocal translocation between ZBTB16 on chromosome 11 and RARA genes on chromosome 17 [t(11;17)(q23;q21)], is rarely observed in acute myeloid leukemia (AML), and accounts for about 1% of retinoic acid receptor-α (RARA) rearrangements. AML with this rare translocation shows unusual bone marrow (BM) morphology, with intermediate aspects between acute promyelocytic leukemia (APL) and AML with maturation. Patients may have a high incidence of disseminated intravascular coagulation at diagnosis, are poorly responsive to all-trans retinoic acid (ATRA) and arsenic tryoxyde, and are reported to have an overall poor prognosis. AIMS: The mutational profile of ZBTB16-RARA rearranged AML has not been described so far. MATERIALS AND METHODS: We performed targeted next-generation sequencing of 24 myeloid genes in BM diagnostic samples from seven ZBTB16-RARA+AML, 103 non-RARA rearranged AML, and 46 APL. The seven ZBTB16-RARA-positive patients were then screened for additional mutations using whole exome sequencing (n = 3) or an extended cancer panel including 409 genes (n = 4). RESULTS: ZBTB16-RARA+AML showed an intermediate number of mutations per patient and involvement of different genes, as compared to APL and other AMLs. In particular, we found a high incidence of ARID1A mutations in ZBTB16-RARA+AML (five of seven cases, 71%). Mutations in ARID2 and SMARCA4, other tumor suppressor genes also belonging to SWI/SNF chromatin remodeling complexes, were also identified in one case (14%). DISCUSSION AND CONCLUSION: Our data suggest the association of mutations of the ARID1A gene and of the other members of the SWI/SNF chromatin remodeling complexes with ZBTB16-RARA+AMLs, where they may support the peculiar disease phenotype.

What's new in the pathogenesis and treatment of therapy-related myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MNs) include diseases onsetting in patients treated with chemo- and/or radiotherapy for a primary cancer, or an autoimmune disorder. Genomic variants, in particular, in familial cancer genes, may play a predisposing role. Recent advances in deep sequencing techniques have shed light on the pathogenesis of t-MNs, identifying clonal hematopoiesis of indeterminate potential (CHIP) as a frequent first step in the multihit model of t-MNs. CHIP is often detectable prior to any cytotoxic treatment, probably setting the fertile genomic background for secondary leukemogenesis. The evolution pattern toward t-MNs is then a complex process, shaped by the type of cancer therapy, the aging process, and the individual exposures, that favor additional hits, such as the acquisition of TP53 mutations and unfavorable karyotype abnormalities. The pathogenesis of t-MNs differs from MN associated with environmental exposure. Indeed, the genetic aberration patterns of MN developing in atomic bomb survivors show few mutations in classical DNA methylation genes, and a high prevalence of 11q and ATM alterations, together with TP53 mutations. Survival in t-MNs is poor. In addition to the biology of t-MNs, the patient's previous disease history and the remission status at t-MN diagnosis are significant factors contributing to unfavorable outcome. New drugs active in secondary leukemias include CPX-351, or venetoclax in combination with hypomethylating agents, monoclonal antibodies as magrolimab, or targeted drugs against pathogenic mutations. Allogeneic stem cell transplantation remains the best currently available therapeutic option with curative intent for fit patients with unfavorable genetic profiles.

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.

In vitro effect of eltrombopag alone and in combination with azacitidine on megakaryopoiesis in patients with myelodysplastic syndrome.

Thrombocytopenia is a severe complication for patients with myelodysplastic syndrome (MDS). Eltrombopag increases platelet count in MDS patients but its combination with azacitidine elicited controversial results. We aimed to quantify the colony forming units of megakaryocytes (CFU-Mk) obtained from CD34+ bone marrow cells isolated from patients with MDS and from healthy donors that were cultured in vitro in the presence or absence of azacitidine and with or without the sequential addition of eltrombopag to the culture medium. CD34+ bone marrow cells from 6 MDS patients and 3 controls were expanded in vitro and cultured for 3 days with or without azacitidine. Subsequently, a CFU-Mk assay was performed in presence or absence of eltrombopag. The addition of eltrombopag in the CFU-Mk assay after mock treatment of CD34+ cells increased the number of CFU-Mk in both controls and patients. On the contrary, using azacitidine pretreated CD34+ cells, eltrombopag minimally increased CFU-Mk in controls and produced heterogeneous response in MDS patients with no change in two patients and CFU-Mk increase in four patients. In vitro CFU-Mk assay suggest that some MDS patients are likely to benefit from the sequential addition of eltrombopag after azacitidine treatment, in the context of a personalized medicine.

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.

Cytotoxicity and Differentiating Effect of the Poly(ADP-Ribose) Polymerase Inhibitor Olaparib in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are highly heterogeneous myeloid diseases, characterized by frequent genetic/chromosomal aberrations. Olaparib is a potent, orally bioavailable poly(ADP-ribose) polymerase 1 (PARP1) inhibitor with acceptable toxicity profile, designed as targeted therapy for DNA repair defective tumors. Here, we investigated olaparib activity in primary cultures of bone marrow mononuclear cells collected from patients with MDS (n = 28). A single treatment with olaparib induced cytotoxic effects in most samples, with median IC50 of 5.4 µM (2.0-24.8 µM), lower than plasma peak concentration reached in vivo. In addition, olaparib induced DNA damage as shown by a high proportion of γH2AX positive cells in samples with low IC50s. Olaparib preferentially killed myeloid cells causing a significant reduction of blasts and promyelocytes, paralleled by an increase in metamyelocytes and mature granulocytes while sparing lymphocytes that are not part of the MDS clone. Consistently, flow cytometry analysis revealed a decrease of CD117+/CD123+ immature progenitors (p < 0.001) and induction of CD11b+/CD16+ (p < 0.001) and CD10+/CD15+ (p < 0.01) neutrophils. Morphological and immunophenotypic changes were associated with a dose-dependent increase of PU.1 and CEBPA transcription factors, which are drivers of granulocytic and monocytic differentiation. Moreover, the combination of olaparib with decitabine resulted in augmented cytotoxic and differentiating effects. Our data suggest that olaparib may have therapeutic potential in MDS patients.

Transcription factors implicated in late megakaryopoiesis as markers of outcome after azacitidine and allogeneic stem cell transplantation in myelodysplastic syndrome.

The hypomethylating agent azacitidine (AZA) is used to treat higher-risk myelodysplastic syndromes (HR-MDS) and elderly patients with low-blast count acute myeloid leukemia (LBC-AML). Platelet recovery is an early predictor of AZA response. We prospectively studied the expression profile of transcription factors, critical for late megakaryopoiesis and changes in their expression after AZA treatment in patients with HR-MDS and LBC-AML enrolled in the BMT-AZA trial (EudraCT number 2010-019673-15). Twenty-five additional patients with low-risk (LR)-MDS were also studied. At the time of diagnosis, GATA2 mRNA levels were significantly higher in MDS as compared to controls, with increasing levels from LR- to HR-MDS/AML. RUNX1 expression was also significantly higher in MDS, as compared to controls, but no differences were found between LR- and HR-MDS. Looking at biomarkers of response, we found that patients AZA responsive had higher basal GATA1 and lower FLI1 expression, compared to those with stable or progressive disease after treatment. Univariate analysis showed that increased GATA2 mRNA expression was associated with a worse overall survival. Our findings suggest that high GATA2 expression is a poor prognostic marker for survival in patients with HR-MDS and LBC-AML treated with azacitidine. Moreover, GATA1 and FLI1 mRNA expression may predict response to AZA treatment.

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.

The Role of Forkhead Box Proteins in Acute Myeloid Leukemia.

Forkhead box (FOX) proteins are a group of transcriptional factors implicated in different cellular functions such as differentiation, proliferation and senescence. A growing number of studies have focused on the relationship between FOX proteins and cancers, particularly hematological neoplasms such as acute myeloid leukemia (AML). FOX proteins are widely involved in AML biology, including leukemogenesis, relapse and drug sensitivity. Here we explore the role of FOX transcription factors in the major AML entities, according to "The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia", and in the context of the most recurrent gene mutations identified in this heterogeneous disease. Moreover, we report the new evidences about the role of FOX proteins in drug sensitivity, mechanisms of chemoresistance, and possible targeting for personalized therapies.