Identification of cells of leukemic stem cell origin with non-canonical regenerative properties.

Despite most acute myeloid leukemia (AML) patients entering remission following chemotherapy, outcomes remain poor due to surviving leukemic cells that contribute to relapse. The nature of these enduring cells is poorly understood. Here, through temporal single-cell transcriptomic characterization of AML hierarchical regeneration in response to chemotherapy, we reveal a cell population: AML regeneration enriched cells (RECs). RECs are defined by CD74/CD68 expression, and although derived from leukemic stem cells (LSCs), are devoid of stem/progenitor capacity. Based on REC in situ proximity to CD34-expressing cells identified using spatial transcriptomics on AML patient bone marrow samples, RECs demonstrate the ability to augment or reduce leukemic regeneration in vivo based on transfusion or depletion, respectively. Furthermore, RECs are prognostic for patient survival as well as predictive of treatment failure in AML cohorts. Our study reveals RECs as a previously unknown functional catalyst of LSC-driven regeneration contributing to the non-canonical framework of AML regeneration.

Leukemic progenitor compartment serves as a prognostic measure of cancer stemness in patients with acute myeloid leukemia.

We systematically investigate functional and molecular measures of stemness in patients with acute myeloid leukemia (AML) using a cohort of 121 individuals. We confirm that the presence of leukemic stem cells (LSCs) detected through in vivo xenograft transplantation is associated with poor survival. However, the measurement of leukemic progenitor cells (LPCs) through in vitro colony-forming assays provides an even stronger predictor of overall and event-free survival. LPCs not only capture patient-specific mutations but also retain serial re-plating ability, demonstrating their biological relevance. Notably, LPC content represents an independent prognostic factor in multivariate analyses including clinical guidelines of risk stratification. Our findings suggest that LPCs provide a robust functional measure of AML, enabling quantitative and rapid assessment of a wide range of patients. This highlights the potential of LPCs as a valuable prognostic factor in AML management.

Chemical genomics reveals targetable programs of human cancers rooted in pluripotency.

Overlapping principles of embryonic and tumor biology have been described, with recent multi-omics campaigns uncovering shared molecular profiles between human pluripotent stem cells (hPSCs) and adult tumors. Here, using a chemical genomic approach, we provide biological evidence that early germ layer fate decisions of hPSCs reveal targets of human cancers. Single-cell deconstruction of hPSCs-defined subsets that share transcriptional patterns with transformed adult tissues. Chemical screening using a unique germ layer specification assay for hPSCs identified drugs that enriched for compounds that selectively suppressed the growth of patient-derived tumors corresponding exclusively to their germ layer origin. Transcriptional response of hPSCs to germ layer inducing drugs could be used to identify targets capable of regulating hPSC specification as well as inhibiting adult tumors. Our study demonstrates properties of adult tumors converge with hPSCs drug induced differentiation in a germ layer specific manner, thereby expanding our understanding of cancer stemness and pluripotency.

Reprogramming of Acute Myeloid Leukemia Patients Cells: Harboring Cancer Mutations Requires Targeting of AML Hierarchy.

Screening of primary patient acute myeloid leukemia (AML) cells is challenging based on intrinsic characteristics of human AML disease and patient-specific conditions required to sustain AML cells in culture. This is further complicated by inter- and intra-patient heterogeneity, and "contaminating" normal cells devoid of molecular AML mutations. Derivation of induced pluripotent stem cells (iPSCs) from human somatic cells has provided approaches for the development of patient-specific models of disease biology and has recently included AML. Although reprogramming patient-derived cancer cells to pluripotency allows for aspects of disease modeling, the major limitation preventing applications and deeper insights using AML-iPSCs is the rarity of success and limited subtypes of AML disease that can be captured by reprogramming to date. Here, we tested and refined methods including de novo, xenografting, naïve versus prime states and prospective isolation for reprogramming AML cells using a total of 22 AML patient samples representing the wide variety of cytogenetic abnormalities. These efforts allowed us to derive genetically matched healthy control (isogenic) lines and capture clones found originally in patients with AML. Using fluorescently activated cell sorting, we revealed that AML reprogramming is linked to the differentiation state of diseased tissue, where use of myeloid marker CD33 compared to the stem cell marker, CD34, reduces reprogramming capture of AML+ clones. Our efforts provide a platform for further optimization of AML-iPSC generation, and a unique library of iPSC derived from patients with AML for detailed cellular and molecular study.

Challenges in Cell Fate Acquisition to Scid-Repopulating Activity from Hemogenic Endothelium of hiPSCs Derived from AML Patients Using Forced Transcription Factor Expression.

The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a "hemogenic endothelium" (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFVCD45-) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.

Targeting SUMOylation dependency in human cancer stem cells through a unique SAE2 motif revealed by chemical genomics.

Natural products (NPs) encompass a rich source of bioactive chemical entities. Here, we used human cancer stem cells (CSCs) in a chemical genomics campaign with NP chemical space to interrogate extracts from diverse strains of actinomycete for anti-cancer properties. We identified a compound (McM25044) capable of selectively inhibiting human CSC function versus normal stem cell counterparts. Biochemical and molecular studies revealed that McM025044 exerts inhibition on human CSCs through the small ubiquitin-like modifier (SUMO) cascade, found to be hyperactive in a variety of human cancers. McM025044 impedes the SUMOylation pathway via direct targeting of the SAE1/2 complex. Treatment of patient-derived CSCs resulted in reduced levels of SUMOylated proteins and suppression of progenitor and stem cell capacity measured in vitro and in vivo. Our study overcomes a barrier in chemically inhibiting oncogenic SUMOylation activity and uncovers a unique role for SAE2 in the biology of human cancers.

Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients.

The aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2+ AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ+), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue.

Phosphorylation state of the histone variant H2A.X controls human stem and progenitor cell fate decisions.

Histone variants (HVs) are a subfamily of epigenetic regulators implicated in embryonic development, but their role in human stem cell fate remains unclear. Here, we reveal that the phosphorylation state of the HV H2A.X (γH2A.X) regulates self-renewal and differentiation of human pluripotent stem cells (hPSCs) and leukemic progenitors. As demonstrated by CRISPR-Cas deletion, H2A.X is essential in maintaining normal hPSC behavior. However, reduced levels of γH2A.X enhances hPSC differentiation toward the hematopoietic lineage with concomitant inhibition of neural development. In contrast, activation and sustained levels of phosphorylated H2A.X enhance hPSC neural fate while suppressing hematopoiesis. This controlled lineage bias correlates to occupancy of γH2A.X at genomic loci associated with ectoderm versus mesoderm specification. Finally, drug modulation of H2A.X phosphorylation overcomes differentiation block of patient-derived leukemic progenitors. Our study demonstrates HVs may serve to regulate pluripotent cell fate and that this biology could be extended to somatic cancer stem cell control.

Human pluripotent stem cells identify molecular targets of trisomy 12 in chronic lymphocytic leukemia patients.

Identifying precise targets of individual cancers remains challenging. Chronic lymphocytic leukemia (CLL) represents the most common adult hematologic malignancy, and trisomy 12 (tri12) represents a quarter of CLL patients. We report that tri12 human pluripotent stem cells (hPSCs) allow for the identification of gene networks and targets specific to tri12, which are controlled by comparative normal PSCs. Identified targets are upregulated in tri12 leukemic cells from a cohort of 159 patients with monoclonal B cell lymphocytosis and CLL. tri12 signaling patterns significantly influence progression-free survival. Actionable targets are identified using high-content drug testing and functionally validated in an additional 44 CLL patient samples. Using xenograft models, interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor is potent and selective against human tri12 CLL versus healthy patient-derived xenografts. Our study uses hPSCs to uncover targets from genetic aberrations and apply them to cancer. These findings provide immediate translational potential as biomarkers and targets for therapeutic intervention.

Identification of Chemotherapy-Induced Leukemic-Regenerating Cells Reveals a Transient Vulnerability of Human AML Recurrence.

Despite successful remission induction, recurrence of acute myeloid leukemia (AML) remains a clinical obstacle thought to be caused by the retention of dormant leukemic stem cells (LSCs). Using chemotherapy-treated AML xenografts and patient samples, we have modeled patient remission and relapse kinetics to reveal that LSCs are effectively depleted via cell-cycle recruitment, leaving the origins of relapse unclear. Post-chemotherapy, in vivo characterization at the onset of disease relapse revealed a unique molecular state of leukemic-regenerating cells (LRCs) responsible for disease re-growth. LRCs are transient, can only be detected in vivo, and are molecularly distinct from therapy-naive LSCs. We demonstrate that LRC features can be used as markers of relapse and are therapeutically targetable to prevent disease recurrence.

A phase 1 trial evaluating thioridazine in combination with cytarabine in patients with acute myeloid leukemia.

We completed a phase 1 dose-escalation trial to evaluate the safety of a dopamine receptor D2 (DRD2) antagonist thioridazine (TDZ), in combination with cytarabine. Thirteen patients 55 years and older with relapsed or refractory acute myeloid leukemia (AML) were enrolled. Oral TDZ was administered at 3 dose levels: 25 mg (n = 6), 50 mg (n = 4), or 100 mg (n = 3) every 6 hours for 21 days. Intermediate-dose cytarabine was administered on days 6 to 10. Dose-limiting toxicities (DLTs) included grade 3 QTc interval prolongation in 1 patient at 25 mg TDZ and neurological events in 2 patients at 100 mg TDZ (gait disturbance, depressed consciousness, and dizziness). At the 50-mg TDZ dose, the sum of circulating DRD2 antagonist levels approached a concentration of 10 µM, a level noted to be selectively active against human AML in vitro. Eleven of 13 patients completed a 5-day lead-in with TDZ, of which 6 received TDZ with hydroxyurea and 5 received TDZ alone. During this period, 8 patients demonstrated a 19% to 55% reduction in blast levels, whereas 3 patients displayed progressive disease. The extent of blast reduction during this 5-day interval was associated with the expression of the putative TDZ target receptor DRD2 on leukemic cells. These preliminary results suggest that DRD2 represents a potential therapeutic target for AML disease. Future studies are required to corroborate these observations, including the use of modified DRD2 antagonists with improved tolerability in AML patients. This trial was registered at www.clinicaltrials.gov as #NCT02096289.

CXCL12/CXCR4 Signaling Enhances Human PSC-Derived Hematopoietic Progenitor Function and Overcomes Early In Vivo Transplantation Failure.

Human pluripotent stem cells (hPSCs) generate hematopoietic progenitor cells (HPCs) but fail to engraft xenograft models used to detect adult/somatic hematopoietic stem cells (HSCs) from donors. Recent progress to derive hPSC-derived HSCs has relied on cell-autonomous forced expression of transcription factors; however, the relationship of bone marrow to transplanted cells remains unknown. Here, we quantified a failure of hPSC-HPCs to survive even 24 hr post transplantation. Across several hPSC-HPC differentiation methodologies, we identified the lack of CXCR4 expression and function. Ectopic CXCR4 conferred CXCL12 ligand-dependent signaling of hPSC-HPCs in biochemical assays and increased migration/chemotaxis, hematopoietic progenitor capacity, and survival and proliferation following in vivo transplantation. This was accompanied by a transcriptional shift of hPSC-HPCs toward somatic/adult sources, but this approach failed to produce long-term HSC xenograft reconstitution. Our results reveal that networks involving CXCR4 should be targeted to generate putative HSCs with in vivo function from hPSCs.

Acute myeloid leukaemia disrupts endogenous myelo-erythropoiesis by compromising the adipocyte bone marrow niche.

Acute myeloid leukaemia (AML) is distinguished by the generation of dysfunctional leukaemic blasts, and patients characteristically suffer from fatal infections and anaemia due to insufficient normal myelo-erythropoiesis. Direct physical crowding of bone marrow (BM) by accumulating leukaemic cells does not fully account for this haematopoietic failure. Here, analyses from AML patients were applied to both in vitro co-culture platforms and in vivo xenograft modelling, revealing that human AML disease specifically disrupts the adipocytic niche in BM. Leukaemic suppression of BM adipocytes led to imbalanced regulation of endogenous haematopoietic stem and progenitor cells, resulting in impaired myelo-erythroid maturation. In vivo administration of PPARγ agonists induced BM adipogenesis, which rescued healthy haematopoietic maturation while repressing leukaemic growth. Our study identifies a previously unappreciated axis between BM adipogenesis and normal myelo-erythroid maturation that is therapeutically accessible to improve symptoms of BM failure in AML via non-cell autonomous targeting of the niche.

Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/ß-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/ß-catenin signaling within CSCs. Disruption of CBP-ß-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

Playing musical chairs with bone marrow transplantation to eliminate leukemia stem cells.

Increasing attention has been focused on the interactions between leukemia cells and their bone marrow (BM) microenvironment. We have recently shown that leukemic stem cells (LSCs) share common BM "niches" with their healthy counterparts. As a result of these shared niche requirements, human LSCs can be mobilized using existing pharmacological agents that facilitate competitive healthy reconstitution, leading to eradication of leukemia during BM transplantation.

Niche displacement of human leukemic stem cells uniquely allows their competitive replacement with healthy HSPCs.

Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is currently the leading strategy to manage acute myeloid leukemia (AML). However, treatment-related morbidity limits the patient generalizability of HSCT use, and the survival of leukemic stem cells (LSCs) within protective areas of the bone marrow (BM) continues to lead to high relapse rates. Despite growing appreciation for the significance of the LSC microenvironment, it has remained unresolved whether LSCs preferentially situate within normal HSC niches or whether their niche requirements are more promiscuous. Here, we provide functional evidence that the spatial localization of phenotypically primitive human AML cells is restricted to niche elements shared with their normal counterparts, and that their intrinsic ability to initiate and retain occupancy of these niches can be rivaled by healthy hematopoietic stem and progenitor cells (HSPCs). When challenged in competitive BM repopulation assays, primary human leukemia-initiating cells (L-ICs) can be consistently outperformed by HSPCs for BM niche occupancy in a cell dose-dependent manner that ultimately compromises long-term L-IC renewal and subsequent leukemia-initiating capacity. The effectiveness of this approach could be demonstrated using cytokine-induced mobilization of established leukemia from the BM that facilitated the replacement of BM niches with transplanted HSPCs. These findings identify a functional vulnerability of primitive leukemia cells, and suggest that clinical development of these novel transplantation techniques should focus on the dissociation of L-IC-niche interactions to improve competitive replacement with healthy HSPCs during HSCT toward increased survival of patients.

Molecular pathways: epigenetic modulation of Wnt-glycogen synthase kinase-3 signaling to target human cancer stem cells.

Aberrant regulation of the canonical Wnt signaling pathway (Wnt-ß-catenin-GSK3 axis) has been a prevalent theme in cancer biology since earlier observations until recent genetic discoveries gleaned from tumor genome sequencing. During the last few decades, a large body of work demonstrated the involvement of the Wnt-ß-catenin-GSK3 signaling axis in the formation and maintenance of cancer stem cells (CSC) responsible for tumor growth in several types of human malignancies. Recent studies have elucidated epigenetic mechanisms that control pluripotency and stemness, and allow a first assessment on how embryonic and normal tissue stem cells are dysregulated in cancer to give rise to CSCs, and how canonical Wnt signaling might be involved. Here, we review emerging concepts highlighting the critical role of epigenetics in CSC development through abnormal canonical Wnt signaling. Finally, we refer to the characterization of novel and powerful inhibitors of chromatin organization machinery that, in turn, restore the Wnt-ß-catenin-GSK3 signaling axis in malignant cells, and describe attempts/relevance to bring these compounds into preclinical and clinical studies.

Bone marrow localization and functional properties of human hematopoietic stem cells.

PURPOSE OF REVIEW: Historically, studies of the hematopoietic stem cell (HSC) microenvironment in bone marrow have focused on the identification of individual supportive cell lineages likely to be responsible for maintaining HSCs in a self-renewing and regenerative state. More recently, awareness has developed regarding the broad and dynamic heterogeneity of nonhematopoietic cells that reside within the bone marrow space. We review recent insights that provide an emerging and complex context for understanding the spatially dependent regulation of HSC functional properties in the bone marrow and the collective inputs of multiple cell types. RECENT FINDINGS: Within the last 18 months, high-resolution imaging, xenograft modeling, and genetic mouse models have afforded innovative methods of detecting and interrogating HSCs with precision at the cellular level. Spatially distinct sites within the bone marrow house functionally divergent HSCs and progenitors, and these different habitats are becoming carefully characterized from a cellular and molecular perspective. This is critical toward understanding how bone marrow microenvironments adapt to accommodate cellular demands for hematopoiesis and how these mechanisms are disrupted in pathological conditions. SUMMARY: The bone marrow is not a continuum but an integrated unit with complex trophic interactions. Emphasis on human data will become necessary as these concepts mature and develop translationally toward changing clinical practices in HSC transplantation and even in the treatment of leukemias.

Regional localization within the bone marrow influences the functional capacity of human HSCs.

Numerous studies have shown that the bone marrow (BM) niche plays a key role in mouse hematopoietic stem cell (HSC) function and involves contributions from a broad array of cell types. However, the composition and role of the human BM HSC niche have not been investigated. Here, using human bone biopsy specimens, we provide evidence of HSC propensity to localize to endosteal regions of the trabecular bone area (TBA). Through functional xenograft transplantation, we found that human HSCs localizing to the TBA have superior regenerative and self-renewal capacity and are molecularly distinct from those localizing to the long bone area (LBA). In addition, osteoblasts in the TBA possess unique characteristics and express a key network of factors that regulate TBA- versus LBA-localized human HSCs in vivo. Our study reveals that BM localization and architecture play a critical role in defining the functional and molecular properties of human HSCs.

Nonhematopoietic cells represent a more rational target of in vivo hedgehog signaling affecting normal or acute myeloid leukemia progenitors.

Recent work has shown that leukemic stem cell self-renewal in chronic myeloid leukemia is dependent on cell-intrinsic hedgehog (Hh) signaling, and early clinical trials suggest that targeting this pathway is also therapeutic in patients with acute myeloid leukemia (AML). In this study, we aimed to better understand Hh signaling in normal hematopoiesis and AML by molecularly and functionally analyzing more than 200 primary human AML patient samples compared with nonleukemic controls. Gene expression analysis indicated that Hh pathway transcripts were similarly regulated in AML and nonleukemic controls, regardless of whether samples were purified based on primitive phenotypes. Consistent with these results, pharmacologic inhibition of Smoothened (SMO) did not preferentially reduce in vitro colony formation of AML versus normal progenitors. Using a unique analytic approach, messenger RNA expression of membrane receptor SMO was found to be unexpectedly rare within all hematopoietic samples analyzed, which is indicative of heterogeneity at the level of Hh signaling machinery. In contrast, abundant SMO expression could be readily detected in the nonhematopoietic fraction of human and murine bone marrow (BM) cells. Our predictions of increased SMO(+) cell frequencies within nonhematopoietic BM fractions were further supported by single-cell protein analyses. Although we did not find support for cell-autonomous sensitivity of AML cells to Hh pathway inhibition, we alternatively suggest that nonhematopoietic BM cells represent an indirect target through which primitive normal and leukemic cells can be modulated. These findings suggest current approaches to applying Hh inhibition should be carefully reevaluated to account for BM niche cell regulation that might be selectively Hh responsive.