Combinatorial BCL2 family expression in acute myeloid leukemia stem cells predicts clinical response to azacitidine/venetoclax

UNLABELLED: The BCL2 inhibitor venetoclax (VEN) in combination with azacitidine (5-AZA) is currently transforming acute myeloid leukemia (AML) therapy. However, there is a lack of clinically relevant biomarkers that predict response to 5-AZA/VEN. Here, we integrated transcriptomic, proteomic, functional, and clinical data to identify predictors of 5-AZA/VEN response. Although cultured monocytic AML cells displayed upfront resistance, monocytic differentiation was not clinically predictive in our patient cohort. We identified leukemic stem cells (LSC) as primary targets of 5-AZA/VEN whose elimination determined the therapy outcome. LSCs of 5-AZA/VEN-refractory patients displayed perturbed apoptotic dependencies. We developed and validated a flow cytometry-based Mediators of apoptosis combinatorial score (MAC-Score) linking the ratio of protein expression of BCL2, BCL-xL, and MCL1 in LSCs. MAC scoring predicts initial response with a positive predictive value of more than 97% associated with increased event-free survival. In summary, combinatorial levels of BCL2 family members in AML-LSCs are a key denominator of response, and MAC scoring reliably predicts patient response to 5-AZA/VEN. SIGNIFICANCE: Venetoclax/azacitidine treatment has become an alternative to standard chemotherapy for patients with AML. However, prediction of response to treatment is hampered by the lack of clinically useful biomarkers. Here, we present easy-to-implement MAC scoring in LSCs as a novel strategy to predict treatment response and facilitate clinical decision-making. This article is highlighted in the In This Issue feature, p. 1275

c-Fos induces chondrogenic tumor formation in immortalized human mesenchymal progenitor cells

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation.This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 to J.G.-C.; and RTICC: RD12/0036/0027 to J.G-C, RD12/0036/0020 to S.M.) and the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C) in Spain. A.A. was supported by Juan de la Cierva program of the Spanish Plan Nacional (MINECO) and Sara Borrell program of the ISCIII/FEDER. A.Al. was supported by the “Miguel Servet” program of the ISCIII/FEDER. We gratefully acknowledge support from Asociación Pablo Ugarte (CIF G86121019) and AFANION (CIF G02223733). The experiments were approved by the appropriate committees.S

Modelling the crosstalk between residual normal haematopoietic stem/progenitor cells, acute myeloid leukaemia and their microenvironment

In acute myeloid leukaemia (AML) the suppression of haematopoietic stem and progenitor cell (HSPC) differentiation and proliferation is a major factor contributing to treatment complications and mortality rates. Previous studies associated an altered microenvironment with dysregulated haematopoiesis but the precise molecular mechanisms are still not fully understood. To mimic AML infiltrated human bone marrow (BM), we utilised our published humanised 3D BM model. Healthy donor MSCs were cultured on a 3D collagen scaffold, co-injected with normal CD34+ HSPCs with AML and transplanted into NSG-SGM3 mice. In analogue, we co-cultured healthy BM MSCs with HSPCs and AML. Both ex vivo and in vivo models replicated a suppression of normal haematopoiesis. Similar to AML patients, cell death was affected minimally but the majority of HSPCs were quiescent and showed an enrichment of serial colony-forming cells and LTC-ICs. Ex vivo, HSPC suppression was largely dependent on the presence of MSCs but not in a direct contact manner. Stroma secretome analysis identified the previously uncharacterized stanniocalcin-1 (STC1). STC1 is exclusively expressed in MSCs and upregulated 2 to 30-fold after AML contact. Supplemented recombinant STC1 significantly enriches for quiescent HSPCs and helps to preserve their functionality, while the proliferation of AML was unchanged. Neutralisation of STC1 in AML scaffolds improved HSPC cell number through increased proliferation. Further transcriptomic interrogation of the MSCs after AML exposure also identified an upregulation of Hypoxia-inducible factor -1 alpha (HIF-1α) target genes and increased stabilisation of HIF-1α. Knockdown of HIF-1α in MSCs not only abrogates secreted STC1 but also improves HSPC proliferation in the presence of AML. In conclusion, the newly developed humanised ex vivo and in vivo models mimic the suppression of HSPCs by AML but are dependent on an altered MSC niche and the HIF-1α-STC1 axis. Inhibition of niche remodelling may be an interesting new approach to improve recovery after AML treatment.Open Acces

Generation of neighbor-labeling cells to study intercellular interactions in vivo

Understanding cell-cell interactions is critical in most, if not all, research fields in biology. Nevertheless, studying intercellular crosstalk in vivo remains a relevant challenge, due mainly to the difficulty in spatially locating the surroundings of particular cells in the tissue. Cherry-niche is a powerful new method that enables cells expressing a fluorescent protein to label their surrounding cells, facilitating their specific isolation from the whole tissue as live cells. We previously applied Cherry-niche in cancer research to study the tumor microenvironment (TME) in metastasis. Here we describe how to generate cancer cells with the ability to label their neighboring cells (within the tumor niche) by transferring a liposoluble fluorescent protein. Live niche cells can be isolated and compared with cells distant from the tumor bulk, using a variety of ex vivo approaches. As previously shown, this system has the potential to identify novel components in the TME and improve our understanding of their local interactions. Importantly, Cherry-niche can also be applied to study potential cell-cell interactions due to in vivo proximity in research fields beyond cancer. This protocol takes 2-3 weeks to generate the labeling cells and 1-2 weeks to test their labeling ability

Clonally resolved single-cell multi-omics identifies routes of cellular differentiation in acute myeloid leukemia

Inter-patient variability and the similarity of healthy and leukemic stem cells (LSCs) have impeded the characterization of LSCs in acute myeloid leukemia (AML) and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. Although residual healthy and preleukemic cells dominated the dormant stem cell compartment, active LSCs resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors constituted a highly aberrant, disease-defining compartment: their gene expression and differentiation state affected both the chemotherapy response and leukemia's ability to differentiate into transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers misregulated specifically in leukemic cells. Taken together, CloneTracer reveals a differentiation landscape that mimics its healthy counterpart and may determine biology and therapy response in AML.ISSN:1934-5909ISSN:1875-977