An arms-race against resistance: leukemic stem cells and lineage plasticity.

Acute myeloid leukemia (AML) therapy is undergoing rapid development, but primary and acquired resistance to therapy complicates the prospect of a durable cure. Recent functional and single-cell multi-omics approaches have greatly expanded our knowledge of the diversity of lineage trajectories in AML settings. AML cells range from undifferentiated stem-like cells to more differentiated myeloid or megakaryocyte/erythroid cells. Current clinically relevant drugs predominantly target the myeloid progenitor lineage, while monocyte- or stem cell-like states can evade current AML treatment and may be targeted in the future with lineage-specific inhibitors. The extent of aberrant lineage plasticity upon therapeutic pressure in AML cells in conjunction with hijacking of normal differentiation pathways is still a poorly understood topic. Insights into the mechanisms of lineage plasticity of AML stem cells could identify both therapy-specific and cross-drug resistance pathways and reveal novel strategies to overcome them.

GPRC5C drives branched-chain amino acid metabolism in leukemogenesis.

Leukemia stem cells (LSCs) share numerous features with healthy hematopoietic stem cells (HSCs). G-protein coupled receptor family C group 5 member C (GPRC5C) is a regulator of HSC dormancy. However, GPRC5C functionality in acute myeloid leukemia (AML) is yet to be determined. Within patient AML cohorts, high GPRC5C levels correlated with poorer survival. Ectopic Gprc5c expression increased AML aggression through the activation of NF-κB, which resulted in an altered metabolic state with increased levels of intracellular branched-chain amino acids (BCAAs). This onco-metabolic profile was reversed upon loss of Gprc5c, which also abrogated the leukemia-initiating potential. Targeting the BCAA transporter SLC7A5 with JPH203 inhibited oxidative phosphorylation and elicited strong antileukemia effects, specifically in mouse and patient AML samples while sparing healthy bone marrow cells. This antileukemia effect was strengthened in the presence of venetoclax and azacitidine. Our results indicate that the GPRC5C-NF-κB-SLC7A5-BCAAs axis is a therapeutic target that can compromise leukemia stem cell function in AML.

The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia.

Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.

Combinatorial BCL2 Family Expression in Acute Myeloid Leukemia Stem Cells Predicts Clinical Response to Azacitidine/Venetoclax.

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.

Protein tyrosine kinase 2b inhibition reverts niche-associated resistance to tyrosine kinase inhibitors in AML.

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

Venetoclax synergizes with gilteritinib in FLT3 wild-type high-risk acute myeloid leukemia by suppressing MCL-1.

BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML.

Antigen presentation safeguards the integrity of the hematopoietic stem cell pool.

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of blood and immune cells. Throughout life, HSPCs acquire oncogenic aberrations that can cause hematological cancers. Although molecular programs maintaining stem cell integrity have been identified, safety mechanisms eliminating malignant HSPCs from the stem cell pool remain poorly characterized. Here, we show that HSPCs constitutively present antigens via major histocompatibility complex class II. The presentation of immunogenic antigens, as occurring during malignant transformation, triggers bidirectional interactions between HSPCs and antigen-specific CD4+ T cells, causing stem cell proliferation, differentiation, and specific exhaustion of aberrant HSPCs. This immunosurveillance mechanism effectively eliminates transformed HSPCs from the hematopoietic system, thereby preventing leukemia onset. Together, our data reveal a bidirectional interaction between HSPCs and CD4+ T cells, demonstrating that HSPCs are not only passive receivers of immunological signals but also actively engage in adaptive immune responses to safeguard the integrity of the stem cell pool.

Future directions in preclinical and translational cancer neuroscience research.

Recent advances in cancer neuroscience necessitate the systematic analysis of neural influences in cancer as potential therapeutic targets in oncology. Here, we outline recommendations for future preclinical and translational research in this field.

Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells.

Hematopoietic stem cells (HSCs) and distinct multipotent progenitor (MPP) populations (MPP1-4) contained within the Lin-Sca-1+c-Kit+ (LSK) compartment have previously been identified using diverse surface-marker panels. Here, we phenotypically define and functionally characterize MPP5 (LSK CD34+CD135-CD48-CD150-). Upon transplantation, MPP5 supports initial emergency myelopoiesis followed by stable contribution to the lymphoid lineage. MPP5, capable of generating MPP1-4 but not HSCs, represents a dynamic and versatile component of the MPP network. To characterize all hematopoietic stem and progenitor cells, we performed RNA-sequencing (RNA-seq) analysis to identify specific transcriptomic landscapes of HSCs and MPP1-5. This was complemented by single-cell RNA-seq analysis of LSK cells to establish the differentiation trajectories from HSCs to MPP1-5. In agreement with functional reconstitution activity, MPP5 is located immediately downstream of HSCs but upstream of the more committed MPP2-4. This study provides a comprehensive analysis of the LSK compartment, focusing on the functional and molecular characteristics of the newly defined MPP5 subset.

Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1.

Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal.

Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations.

The exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, because multiple cell types have been reported to physically associate with HSCs. In this study, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components in 152 full-bone sections from different bone types and 3 HSC reporter lines. We found adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes, and different combinations of those populations, but not proximal to bone, adipocyte, periarteriolar, or Schwann cells. Despite microanatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs had similar distributions. Importantly, their microenvironmental localizations were not different from those of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells both had indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially located close to Cxcl12 stroma and farther from sinusoids/megakaryocytes. We expect our study to help resolve existing confusion regarding the exact localization of different HSC types, their physical association with described BM populations, and their tissue-wide combinations.

Clinically Actionable Strategies for Studying Neural Influences in Cancer.

Neuro-glial activation is a recently identified hallmark of growing cancers. Targeting tumor hyperinnervation in preclinical and small clinical trials has yielded promising antitumor effects, highlighting the need of systematic analysis of neural influences in cancer (NIC). Here, we outline the strategies translating these findings from bench to the clinic.

Differential Alternative Polyadenylation Landscapes Mediate Hematopoietic Stem Cell Activation and Regulate Glutamine Metabolism.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3' untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies remains elusive. Here, we first demonstrate the requirement of the APA regulator Pabpn1 for hematopoietic stem cell (HSC) function. We then determine the genome-wide APA landscape (APAome) of HSCs and progenitors by performing low-input 3' sequencing paired with bioinformatic pipelines. This reveals transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation. Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching by Nudt21. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal, behavior, and commitment.

Eosinophil accumulation predicts response to melanoma treatment with immune checkpoint inhibitors.

Eosinophils have been identified as a prognostic marker in immunotherapy of melanoma and suggested to contribute to anti-tumor host defense. However, the influence of immune checkpoint inhibitors (ICI) on the eosinophil population is poorly studied. Here, we applied routine laboratory tests, multicolor flow cytometry, RNA microarray analysis, and bio-plex assay to analyze circulating eosinophils and related serum inflammatory factors in 32 patients treated with pembrolizumab or the combination of nivolumab and ipilimumab. We demonstrated that clinical responses to ICI treatment were associated with an eosinophil accumulation in the peripheral blood. Moreover, immunotherapy led to the alteration of the eosinophil genetic and activation profile. Elevated serum concentrations of IL-16 during ICI treatment were found to be associated with increased frequencies of eosinophils in the peripheral blood. Using immunohistochemistry, we observed an enhanced eosinophil degranulation and a positive correlation between eosinophil and CD8+ T cell infiltration of tumor tissues from melanoma patients treated with ICI. Our findings highlight additional mechanisms of ICI effects and suggest the level of eosinophils as a novel predictive marker for melanoma patients who may benefit from this immunotherapy.

The long non-coding RNA Meg3 is dispensable for hematopoietic stem cells.

The long non-coding RNA (lncRNA) Maternally Expressed Gene 3 (Meg3) is encoded within the imprinted Dlk1-Meg3 gene locus and is only maternally expressed. Meg3 has been shown to play an important role in the regulation of cellular proliferation and functions as a tumor suppressor in numerous tissues. Meg3 is highly expressed in mouse adult hematopoietic stem cells (HSCs) and strongly down-regulated in early progenitors. To address its functional role in HSCs, we used MxCre to conditionally delete Meg3 in the adult bone marrow of Meg3mat-flox/pat-wt mice. We performed extensive in vitro and in vivo analyses of mice carrying a Meg3 deficient blood system, but neither observed impaired hematopoiesis during homeostatic conditions nor upon serial transplantation. Furthermore, we analyzed VavCre Meg3mat-flox/pat-wt mice, in which Meg3 was deleted in the embryonic hematopoietic system and unexpectedly this did neither generate any hematopoietic defects. In response to interferon-mediated stimulation, Meg3 deficient adult HSCs responded highly similar compared to controls. Taken together, we report the finding, that the highly expressed imprinted lncRNA Meg3 is dispensable for the function of HSCs during homeostasis and in response to stress mediators as well as for serial reconstitution of the blood system in vivo.

Bone Marrow Myeloid Cells Regulate Myeloid-Biased Hematopoietic Stem Cells via a Histamine-Dependent Feedback Loop.

Myeloid-biased hematopoietic stem cells (MB-HSCs) play critical roles in recovery from injury, but little is known about how they are regulated within the bone marrow niche. Here we describe an auto-/paracrine physiologic circuit that controls quiescence of MB-HSCs and hematopoietic progenitors marked by histidine decarboxylase (Hdc). Committed Hdc+ myeloid cells lie in close anatomical proximity to MB-HSCs and produce histamine, which activates the H2 receptor on MB-HSCs to promote their quiescence and self-renewal. Depleting histamine-producing cells enforces cell cycle entry, induces loss of serial transplant capacity, and sensitizes animals to chemotherapeutic injury. Increasing demand for myeloid cells via lipopolysaccharide (LPS) treatment specifically recruits MB-HSCs and progenitors into the cell cycle; cycling MB-HSCs fail to revert into quiescence in the absence of histamine feedback, leading to their depletion, while an H2 agonist protects MB-HSCs from depletion after sepsis. Thus, histamine couples lineage-specific physiological demands to intrinsically primed MB-HSCs to enforce homeostasis.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing.

Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation.

Transcriptome-wide profiling and posttranscriptional analysis of hematopoietic stem/progenitor cell differentiation toward myeloid commitment.

Hematopoietic stem cells possess lifelong self-renewal activity and generate multipotent progenitors that differentiate into lineage-committed and subsequently mature cells. We present a comparative transcriptome analysis of ex vivo isolated mouse multipotent hematopoietic stem/progenitor cells (Lin(neg)SCA-1(+)c-KIT(+)) and myeloid committed precursors (Lin(neg)SCA-1(neg)c-KIT(+)). Our data display dynamic transcriptional networks and identify a stem/progenitor gene expression pattern that is characterized by cell adhesion and immune response components including kallikrein-related proteases. We identify 498 expressed lncRNAs, which are potential regulators of multipotency or lineage commitment. By integrating these transcriptome with our recently reported proteome data, we found evidence for posttranscriptional regulation of processes including metabolism and response to oxidative stress. Finally, our study identifies a high number of genes with transcript isoform regulation upon lineage commitment. This in-depth molecular analysis outlines the enormous complexity of expressed coding and noncoding RNAs and posttranscriptional regulation during the early differentiation steps of hematopoietic stem cells toward the myeloid lineage.