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Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell–cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but >30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

Background.Measurable residual disease (MRD) is a key parameter to evaluate response to cytotoxic treatment in leukemia patients and, thus, to guide therapeutic strategies. In fact, the identification of persisting leukemia cells after chemotherapy is associated with an increased risk of disease recurrence, and many patients with acute myeloid leukemia (AML) experiencing relapse have a fatal outcome. Many somatic genetic aberrations are under investigation for assessing molecular quantitative MRD (qMRD). However, while flow cytometry is already recognized as useful in measuring residual disease levels, the impact of qMRD monitoring is still debated.

In acute myeloid leukemia (AML), the assessment of post-induction minimal residual disease (MRD) is largely utilized for choosing post-remission therapies aimed at maintaining complete remission (CR) and preventing relapse. This latter is still the major cause of treatment failure in pediatric AML, and even if several efforts have been spent to validate MRD as a prognostic marker, numerous studies demonstrated that MRD negativity cannot be considered a completely reliable surrogate biomarker predicting outcome, since it does not exclude a relapse. The current interpretation is that disease relapse is due to mechanisms leading to therapy resistance mainly depending on driver chimeric or oncogenic protein-coding genes, which are monitored during treatment, and does not consider that chemotherapy resistance may arise from other genetic markers, phenomenon linked to methylation and non-coding RNAs genomic pressure. We, thus, hypothesized that other markers need to be explored to re-interpret leukemia progression. We showed an overall hyper-expression of the lncRNA BALR2in 132 de novoAML bone marrow samples collected at diagnosis and analyzed the gene expression profile (GEP) of 58 cases. By unsupervised clustering analysis, we produced important advances in identifying BALR2as a robust novel molecular marker of a new subgroup of AML characterized by a high rate of resistance to induction therapy, independently from the genetic lesions detected at diagnosis and any other prognostic clinical and genetic features. We demonstrated in vitrothat BALR2has a direct role in controlling bi-directionally its own and of its neighbor gene CDK6promoter activity. This latter finding of high CDK6expression was shown to sustain its complex with RUNX1 in order to inhibit RUNX1 binding to its target promoters, thus preventing the process of hematopoietic differentiation progression. To support BALR2as a new proto-oncogene involved in the control of the myeloid differentiation program, we ranked the genes across the expression profile obtaining a signature of 337 transcripts able to cluster CD34+ human stem cell precursors (HSCPs) separately from more mature CD14+ cells. These in silicofindings were validated in vitroby showing that, after BALR2depletion, CD34+ cells had a skewed myeloid differentiation. Furthermore, we found that AML differentiation toward mature myeloid cells with increased phagocytic capacity was obtained through BALR2level reduction, and enhanced by combinatorial differentiation stimuli. Our findings attribute a distinct role to BALR2in the block of myeloid stem cell differentiation occurring during leukemogenesis. At the same time, we interrogated GEP ontology, finding that enrichments of genes involved in mitochondrial synthesis pathways were significantly correlated to patients with highest BALR2levels, and confirmed the same mitochondriogenesis profile in the immature CD34+ HSCPs. We moved to deconvolute this feature and demonstrated that BALR2, by controlling mitochondria gene balance, was directly controlling the mitochondrial mass, which dramatically decreased after BALR2silencing, this supporting the hypothesis that BALR2would maintain mitochondrial functions to confer AML resistance to cytotoxicity. Consistently with this line of reasoning, we inhibited mitochondria by tigecycline, demonstrating that its activity was dramatically strengthened in BALR2depleted cells, when used either alone or in combination with cytosine-arabinoside (Ara-C). Concomitantly, tigecycline treatment in BALR2silenced AML cells reduced mitochondria depolarization, and increased the number of differentiated M-CFU colonies formation, confirming that BALR2,together with CDK6,forms novel transcriptional networks to create a circuit able to impair myeloid differentiation and to lower chemo-sensitivity in AML. We speculate that a novel therapeutic window of mitochondrial targeting in defined AML subgroups, identified through assessment of BALR2levels at diagnosis or persistent MRD levels, could be envisaged to optimize the outcome of childhood AML.

Chemotherapy still remains the pillar of treatment of children with AML, a disease in which refinements in diagnostic approaches, minimal residual disease monitoring, and patient stratification have resulted into remarkable progresses during the past decade. However, most of the recently tested, novel anti-leukemia agents failed during pre-clinical and clinical validation phases, and one main limit in AML field is the inappropriateness of current preclinical models used to study drug efficacy, this jeopardizing the advance of phase II and III clinical trials, especially for children. In light of this consideration, we aimed at creating novel robust in vitroand in vivoapproaches to discover or to re-assess alternative treatments to improve the portfolio of agents active in childhood AML. For this purpose, we developed new protocols for long-term 3D-AML cultures to perform more predictable high throughput drug screening in vitro, and, once identified the best compounds, to create new pre-clinical in vivomodels. We set up the bone marrow (BM) endosteal niche by using a biomimetic 3D structure, made up of engineered hydroxyapatite and collagen I, where we seeded mesenchymal stromal cells derived either from AML patients (AML-MSCs) or from healthy BM donors (h-MSCs), together with osteoblasts, endothelial cells and finally AML blasts. We studied AML cell proliferation and clonogenicity cultured in 3D. We obtained results from twenty 3D long-term cultures of different primary AML, confirming blast proliferation up to 21 days. Clonogenic potential and immunophenotype preservation of the original AML blasts was also documented. At the same time, we compared AML-MSCs with h-MSCs, finding that AML-MSCs exhibited a higher proliferation rate (40% increase proliferation at 72 and 96 hours, p<0.001), and commitment to osteogenic differentiation, this latter occurring after 7 days with respect to 21 days of h-MSCs (p<0.01). To better characterize AML-MSCs features supporting AML cell growth, we cultured AML-MSCs and h-MSCs in an inflammatory environment (IL1β, IL6 and TNFα), observing that AML-MSCs did not exert anti-inflammatory activity by HUVEC tube formation assay (n=6, p<0.001). This latter finding was supported by a peculiar secretome profile defined by mass-spectrometry revealing factors as SERPINE1, CHI3L1, TIMP1, and PTX3 being differentially secreted. Thus, a drug targeting of AML-MSCs would be desirable, and we performed a screening of 480 compounds. This screening identified 17/480 active compounds capable of reducing AML-MSCs proliferation without toxicity over h-MSCs and AML blasts. We identified one main compound able to selectively reduce AML-MSCs proliferation, that, when combined to novel therapeutic agents for AML, such as the Quizartinib, I-BET inhibitor and Dasatinib in the 3D cultures, showed a synergistic effect (CI=0.5, p<0.05) towards FLT3-ITD, MLL-rearranged or c-KITmutated AML. We then proceeded with two pilot studies in order to define the use of this 3D-AML cultures in in vivosetting. We implanted these organoids in the back of NSG mice and monitored leukemia engraftment in the scaffolds, as well as AML dissemination in peripheral blood. We documented the niche being vascularized and well organized by immunohistochemical staining for mCD31, hCD45 and hOPN, whereas we had a low rate of success in AML dissemination out of the niche (1 AML out of 14 implanted in 15 months). On the contrary, we observed that AML cells proliferated in the 3D up to 9 months after implantation, suggesting that this system is more suitable for an easy and quick in locodrug testing. Thus, we implanted AML, after being transduced with luciferase, in the 3D niche in NSG mice and monitored luciferase activity during intra-peritoneal drug treatments. We evaluated some new and old compounds known to target leukemia cells and documented a significant reduction of luciferase in the 3D when a drug was active, supporting our 3D scaffolds as a novel useful in vivosystem to screen selected drugs before the prioritization of the best one to be used in a pre-clinical setting in PDXs. In conclusion, our data support the possibility to work with long-term 3D cultures of AML in vitroto identify new drugs, and we attribute to AML-MSCs a crucial supportive role to be further considered in in vivosettings for novel combo strategies.Finally, these findings could help test new compounds to be validated in future clinical trials.

Purpose:Non-coding RNA (ncRNAs) genes are at least as frequent as protein-coding genes in the human genome; however, our knowledge on their function is still preliminary. Long ncRNAs are arbitrarily defined as transcripts that are over 200 nucleotides in length, and demonstrate great heterogeneity in their molecular mechanisms. Their emerging role as new players in cancer development and therapy response is widely supported by several scientific reports, and we recently found the LncRNA BALR-2 expression significantly correlated with a decreased response to prednisone treatment in B-cell precursor acute lymphoblastic leukemia (Bcp_ALL). Given its association with mixed lineage leukemia (MLL) rearrangements in Bcp-ALL, we investigated BALR-2 involvement in acute myeloid leukemia (AML) leukemogenic pathways.

Introduction.The core binding factor rearrangements, inv(16)(p13;1q22), t(16;16)(p13;q22) and t(8;21)(q22;q22), are among the most frequent cytogenetic abnormalities found in children with acute myeloid leukemia (AML). They are considered mutations associated with good responsiveness to chemotherapy and favorable patient outcome. Despite this, in the AIEOP LAM2002 trial, we found that t(8;21) RUNX1-RUNX1T1-rearranged patients had an unexpectedly high cumulative incidence of relapse (30%), which was comparable to that of patients considered at higher risk of treatment failure in light of recurrent molecular/cytogenetic alterations or levels of minimal residual disease at the end of induction therapy. Thus, we aimed at re-evaluating at both genetic and epigenetic level samples of t(8;21) AML cases collected at diagnosis addressing the hypothesis whether the methylation process may influence response to treatment and contribute to further stratify patients carrying the same genetic lesion.

Purpose.While most of the recurrent molecular marker of AML have been already discovered and used for risk stratification in clinical protocols, post-treatment monitoring of these abnormalities can be useful in the clinical management of patients. In particular, monitoring of minimal residual disease (MRD), largely used in acute lymphoblastic leukemia, is gaining popularity also in AML, in the perspective of optimizing risk stratification of patients in terms of subsequent clinical relapse. While MRD monitoring in AML is mainly based on flow-cytometry approaches, molecular MRD measurements are not currently routinely used for taking clinical decision. Here we examine if the molecular MRD and Allelic Ratio (AR) levels monitoring may influence the survival of FLT3internal tandem duplication (ITD)-mutated AML patients.