Resistant Leukemia Stem Cells Research Articles

Advances in pathogenesis research and challenges in treatment development for acute myeloid leukemia.

Acute myeloid leukemia (AML) develops when hematopoietic stem cells acquire chromosomal and genetic abnormalities, transforming into leukemia stem cells (LSCs) and further gaining driver mutations. Advances in genomic analysis have identified numerous new gene mutations involved in AML development. Recent research has shown that individuals with germline mutations in genes like DDX41 and CEBPA develop AML upon acquiring additional somatic mutations, and the latest WHO classification separates AML with such mutations into distinct disease groups. LSCs are regulated by different metabolic processes than normal stem cells, contributing to drug resistance and relapse. LSCs rely on oxidative phosphorylation (OXPHOS) metabolism for energy production, and venetoclax inhibits this process, affecting LSCs. Resistant LSCs show enhanced glycolysis, which suggests that targeting both OXPHOS and glycolysis is crucial. While targeted therapies like FLT3, BCL-2, and IDH inhibitors have shown efficacy, resistance remains an issue, highlighting the need for new treatment strategies. CAR-T cell therapy is an emerging immunotherapy that shows particular promise for targeting CD123 and CLL-1, with acceptable toxicity. Future developments in CAR-T cell therapy and other immunotherapies are anticipated to improve AML treatment outcomes.

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

CircFAM193B interaction with PRMT6 regulates AML leukemia stem cells chemoresistance through altering the oxidative metabolism and lipid peroxidation.

Most forms of chemotherapy for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), as their underlying mechanisms remain unclear. Here, we have identified circFAM193B, which regulates the redox biology of LSCs and is associated with unfavorable outcomes in AML patients. In vitro and in vivo assays suggested that circFAM193B significantly inhibits LSCs chemotherapy resistance and AML progression. Knockdown circFAM193B enhances mitochondrial OXPHOS function and inhibits the accumulation of reactive oxygen species and lipid peroxidation mediated by chemotherapy, which protects AML cells from oxidative stress-induced cell death. Mechanistically, circFAM193B physically interacts with arginine methyltransferase PRMT6 catalytic domain and enhances the transcription efficiency of key lipid peroxidation factor ALOX15 by decreasing H3R2me2a modification. In summary, we have identified circFAM193B was downregulated in LSCs to promote the survival of LSC by modulating energy metabolism and the redox balance in the postchemotherapy persistence of LSC. Our studies provide a conceptual advance and biological insights regarding the drug resistance of LSCs via circRNA mediated PRMT6-deposited methylarginine signaling.

Ferritin-mediated mitochondrial iron homeostasis is essential for the survival of hematopoietic stem cells and leukemic stem cells.

Iron metabolism plays a crucial role in cell viability, but its relationship with adult stem cells and cancer stem cells is not fully understood. The ferritin complex, responsible for intracellular iron storage, is important in this process. We report that conditional deletion of ferritin heavy chain 1 (Fth1) in the hematopoietic system reduced the number and repopulation capacity of hematopoietic stem cells (HSCs). These effects were associated with a decrease in cellular iron level, leading to impaired mitochondrial function and the initiation of apoptosis. Iron supplementation, antioxidant, and apoptosis inhibitors reversed the reduced cell viability of Fth1-deleted hematopoietic stem and progenitor cells (HSPCs). Importantly, leukemic stem cells (LSCs) derived from MLL-AF9-induced acute myeloid leukemia (AML) mice exhibited reduced Fth1 expression, rendering them more susceptible to apoptosis induced by the iron chelation compared to normal HSPCs. Modulating FTH1 expression using mono-methyl fumarate increased LSCs resistance to iron chelator-induced apoptosis. Additionally, iron supplementation, antioxidant, and apoptosis inhibitors protected LSCs from iron chelator-induced cell death. Fth1 deletion also extended the survival of AML mice. These findings unveil a novel mechanism by which ferritin-mediated iron homeostasis regulates the survival of both HSCs and LSCs, suggesting potential therapeutic strategies for blood cancer with iron dysregulation.

Nanoparticle-encapsulated retinoic acid for the modulation of bone marrow hematopoietic stem cell niche

More effective approaches are needed in the treatment of blood cancers, in particular acute myeloid leukemia (AML), that are able to eliminate resistant leukemia stem cells (LSCs) at the bone marrow (BM), after a chemotherapy session, and then enhance hematopoietic stem cell (HSC) engraftment for the re-establishment of the HSC compartment. Here, we investigate whether light-activatable nanoparticles (NPs) encapsulating all-trans-retinoic acid (RA+NPs) could solve both problems. Our in vitro results show that mouse AML cells transfected with RA+NPs differentiate towards antitumoral M1 macrophages through RIG.1 and OASL gene expression. Our in vivo results further show that mouse AML cells transfected with RA+NPs home at the BM after transplantation in an AML mouse model. The photo-disassembly of the NPs within the grafted cells by a blue laser enables their differentiation towards a macrophage lineage. This macrophage activation seems to have systemic anti-leukemic effect within the BM, with a significant reduction of leukemic cells in all BM compartments, of animals treated with RA+NPs, when compared with animals treated with empty NPs. In a separate group of experiments, we show for the first time that normal HSCs transfected with RA+NPs show superior engraftment at the BM niche than cells without treatment or treated with empty NPs. This is the first time that the activity of RA is tested in terms of long-term hematopoietic reconstitution after transplant using an in situ activation approach without any exogenous priming or genetic conditioning of the transplanted cells. Overall, the approach documented here has the potential to improve consolidation therapy in AML since it allows a dual intervention in the BM niche: to tackle resistant leukemia and improve HSC engraftment at the same time.

Linking DNA Damage Response to Ferroptosis for Leukemia Stem Cell Eradication

Relapse in acute myeloid leukemia (AML) is associated with an unfavorable prognosis, primarily due to the poor response to conventional chemotherapy, which has shown disappointing overall outcomes over the past 30 years. The main culprits behind leukemogenesis and therapy resistance are leukemia stem cells (LSCs), characterized by their unique features of self-renewal and evasion from cell death. In our recent work, we have identified a critical self-renewal pathway, RSPO3-LGR4/p-PKAc, which exhibits aberrant activation in relapsed or refractory AML (Salik et al. Cancer Cell, 2020). Blocking this pathway holds the potential to eradicate resistant LSCs and treat relapsed AML effectively. In this study, our data directly link the self-renewal pathway to ferroptosis resistance through activation of antioxidant defense, which confers protection on AML LSCs against detrimental effects of oxidative stress. We also found that aberrant activation of RSPO3-LGR4/p-PKAc suppressed the DNA damage response (DDR) pathway consequently leading to genomic instability, DNA repair defects, increased stemness and AML aggressiveness. Our whole-genome sequencing and knockout mouse studies showed that deletion of the key DDR gene caused substantial mutations on multiple pathways including DNA damage repair (e.g., Slf1) and self-renewal (e.g., Hox cluster genes), contributing to a more aggressive and therapy-resistant phenotype in AML initiation and progression. Single cell multi-omics sequencing and CRISPR/Cas9 knockout mouse models uncovered essential antioxidant enzymes as downstream targets of DDR pathway inhibition in AML patient-derived LSCs. These antioxidant enzymes induced by DDR pathway inhibition had protective effects against oxidative attack due to the ability to decompose reactive oxygen species (ROS) and detoxify excess iron. ShRNA-mediated stable knockdown of antioxidant enzymes impaired LSCs by increasing accumulation of iron and ROS and sensitized LSCs to ferroptosis (an iron-dependent and ROS-reliant cell death). Our analysis of AML dataset from TCGA (The Cancer Genome Atlas, N Engl J Med 2013) displayed a positive correlation of high expression of antioxidant genes with poor prognosis in a cohort of 172 AML patients (p<0.019), indicating the clinical importance of antioxidant dysregulation in cancer. Collectively, we report here a functional relationship of DDR pathway inhibition and antioxidant defense mechanism that is regulated by RSPO3-LGR4/p-PKAc self-renewal pathway and contributes to ferroptosis resistance in AML LSCs. Targeting essential pathway components enables LSCs to become sensitive to oxidative damage and ferroptosis and perturbs leukemia progression, presenting a promising new therapeutic approach for relapsed or refractory AML.

Targeting Mitochondrial Calcium Uptake to Eradicate Venetoclax-Resistant Acute Myeloid Leukemia Stem Cells

Introduction Previously, we demonstrated that human leukemia stem cells (LSCs) are preferentially reliant on oxidative phosphorylation (OXPHOS) for survival. Furthermore, inhibition of BCL2 with venetoclax (ven) acts to suppress OXPHOS, leading to the eradication of LSCs. While the activity of ven in targeting LSCs is well-established, nearly all patients eventually develop disease that is resistant to treatment. Thus, in the present study, we sought to better understand the biological properties of ven-resistant LSCs. We reasoned that the expression of known BCL2 interacting proteins in ven-sensitive (ven-S) vs. ven-resistant (ven-R) would provide insights on resistance mechanisms. Initial findings showed that one of the most prevalent molecular features observable in ven resistant LSCs is up-regulation of calcium signaling. As calcium is a critical modulator of multiple enzymes involved in OXPHOS, we hypothesized that the maintenance of high mitochondrial calcium could contribute to circumventing the effects of ven treatment. Thus, the present study was designed to test the functional role of calcium in ven-R LSCs and to determine whether targeting mitochondrial calcium transport represents a therapeutic vulnerability. Methods CITE-Seq analysis was performed on 25 AML patient specimens and analyzed using TotalVI. All studies were conducted in primary human AML specimens with LSCs defined by a ROS-low phenotype. Mitochondrial calcium measurements were done using Rhod2-AM. OXPHOS activity was measured through seahorse and enzyme activity assays. Genetic perturbations were done using siRNA via electroporation and confirmed with western blot. LSC function was assessed by colony formation assays and PDX experiments. PDX experiments were also used to determine efficacy of tumor reduction in ven-R AML samples. Results To identify unique features of ven resistant LSCs, we performed single cell RNA-seq on known ven-S vs. ven-R specimens. As shown in Fig 1, resistant LSCs had higher expression of genes involved in calcium transport into the mitochondria, which was reflected by increased steady-state mitochondrial calcium levels compared to ven-S LSCs. Thus, we investigated targeting of the calcium uniporter, MCU, as a means to reduce mitochondrial calcium levels. Treatment of primary LSCs with pharmacological MCU inhibitors (Ru265, MCUi4), as well as genetic inhibition of MCU with siRNAs, led to decreased mitochondrial calcium levels, suppression of calcium dependent TCA cycle dehydrogenase activity, reduced OXPHOS activity, and impaired LSC function as measured by colony assays (CFU) and engraftment in immune deficient mice. Importantly, MCU inhibition did not significantly impair normal stem/progenitor cells. To develop a clinically relevant system, we investigated bio-available agents known to inhibit MCU. Intriguingly, the only known FDA-approved direct MCU inhibitor is the chemotherapy agent, mitoxantrone (mitox). We performed a series of studies to assess the impact of mitox on ven-R LSCs. Our findings demonstrate that venetoclax resistant LSCs are exquisitely sensitive to mitox. In CFU assays, doses as low 1.0nM show strong inhibitory activity. Further, treatment of immune deficient mice bearing human xenografts (Fig 2), showed significant reduction of bulk disease in primary mice, and strong suppression of LSCs in secondary transplants. Notably, DNA damage was not present in LSCs treated with mitox as measured by gamma H2AX staining. Additionally, parallel studies with the related agents, doxorubicin and etoposide, showed no inhibition of CFU, mitochondrial calcium, or OXPHOS activity. Lastly, no discernable effect on normal hematopoietic stem cell activity was evident. Conclusions Our findings suggest that ven-R LSCs have adapted to their unique cellular demands by increasing basal mitochondrial calcium levels to drive OXPHOS. Based on this finding, we propose that inhibition of mitochondrial calcium uptake impairs multiple enzymes required for OXPHOS, which in turns leads to eradication of LSCs. Importantly, we also show that the well-established chemotherapy agent mitoxantrone can effectively inhibit MCU at nanomolar concentrations and is a highly potent against ven-R LSCs. We are currently working to establish a clinical trial in which low-dose mitox will be deployed to augment ven-based therapy.

Targeting BRD4 to Counteract Cytokine-Induced Expression of PD-L1 and PD-L2 on Vascular, Osteoblastic and Stromal Niche Cells

The stem cell niche plays a crucial role in promoting the expansion of leukemic stem cells (LSC) and in mediating resistance of LSC against various anti-neoplastic drugs in different types of leukemias. Recent data suggest that targeting niche cells may be an effective approach to overcome niche-mediated drug resistance of LSC. However, niche cells themselves are considered to be often resistant against therapeutic intervention. This may especially be relevant in those myeloid neoplasms where niche cell expansion is a pathognomonic feature and the resulting neo-angiogenesis, bone marrow fibrosis and/or sclerosis are important prognostic variables. We and others have recently shown that several of the anti-neoplastic drugs used to treat myeloid leukemias can also counteract niche cell expansion. So far, however, little is known about mechanisms underlying resistance of niche cells against anti-neoplastic drugs. Recent studies have shown that certain immune checkpoint antigens are overexpressed in cancer cells and/or their microenvironment and that these checkpoint antigens contribute to drug resistance. Other studies have shown that checkpoint molecules, including PD-L1 and CD47, are displayed by LSC in acute myeloid leukemia, chronic myeloid leukemia and other myeloid neoplasms. We asked whether niche-related cells also display these checkpoint molecules. Furthermore, we screened for cytokines that promote expression of checkpoint antigens on niche cells and asked for drugs that counteract growth and cytokine-induced checkpoint expression in these cells. The following niche cells were examined: primary human umbilical vein endothelial cells (HUVEC), the human endothelial cell line HMEC-1, the human osteoblast-like cell line CAL-72, primary osteoblasts, and the human stromal cell line HS5. Expression of the checkpoint antigens PD-L1, PD-L2, PD1, CD28, CD47, CD80, CD83, CD86 and TIM-3 was determined by flow cytometry. All niche cells were found to constitutively express PD-L1, PD-L2, and CD47. The other checkpoint antigens were expressed at low or undetectable levels in these cells. Expression of PD-L1, PD-L2 and CD47 in niche cells was confirmed by quantitative PCR and by immunocytochemistry. Of all cytokines tested (n=12), interferon-alpha (IFN-A; effective range 50-500 ng/ml) and interferon-gamma (IFN-G; 10-500 ng/ml) were found to promote surface expression of PD-L1 and PD-L2 in HMEC-1, CAL72, and HS5 cells in our immunocytochemical and flow cytometry experiments. Moreover, we found that tumor necrosis factor-alpha (TNF-A; 100 ng/ml) enhances IFN-G-induced expression of PD-L1 and PD-L2 in HMEC-1, CAL-72 and HS5 cells. The BRD4-targeting drug JQ1 and the BRD4 degraders dBET1 and dBET6 suppressed cytokine-induced upregulation of surface expression of PD-L1 and PD-L2 in HMEC-1, HUVEC, CAL-72 and HS5 cells, suggesting a role for BRD4 and MYC in checkpoint regulation. In all cell lines examined, dBET6 was found to be a more effective drug compared to JQ1 and dBET1 (IFN-G-induced expression of PD-L1 in: HMEC-1: IC 50 for JQ1 0.5 µM vs dBET1 1 µM vs dBET6 0.05 µM; CAL-72: IC 50 for JQ1 >0.5 µM vs dBET1 1 µM vs dBET6 <0.05 µM). Together, we show that endothelial cells, osteoblasts and stromal cells display PD-L1, PD-L2 and CD47 in a constitutive manner. Our data also show that the pro-inflammatory cytokines IFN-G and IFN-A augment expression of PD-L1 and PD-L2 in these cells, and that TNF-A can further enhance IFN-A/IFN-G-induced expression of PD-L1 and PD-L2 in niche cells. In addition, we show that the BRD4-targeting drugs JQ1, dBET1 and dBET6 suppress cytokine-induced PD-L1 and PD-L2 expression on niche cells. Whether checkpoint expression in niche cells is associated with drug resistance in myeloid neoplasms and whether drug-induced suppression of checkpoint expression in niche cells can overcome resistance remains unknown.

A Deeply Quiescent Subset of CML LSC depend on FAO yet Avoid Deleterious ROS by Suppressing Mitochondrial Complex I.

Disease relapse and therapy resistance remain serious impediments to treating cancer. Leukemia stem cells (LSC) are therapy resistant and the cause of relapse. A state of deep quiescence appears to enable cancer stem cells (CSC) to acquire new somatic mutations essential for disease progression and therapy resistance. Both normal hematopoietic stem cells (HSC) and LSC share many common features, thereby complicating the safe elimination of LSC. A recent study demonstrated that long lived normal oocytes exist without mitochondrial complex I (MC-1), expressing it in a developmentally regulated fashion, thereby mitigating their vulnerability to ROS. Quiescent CSC rely on mitochondrial FAO, without complex I expression, thereby avoiding the generation of damaging ROS, similar to long lived normal human stem cells. A deeper understanding of the biology of therapy resistance is important for the development of optimal strategies to attain complete leukemia cures. Here, using scRNA-sequencing and ATAC-seq on primary chronic myelogenous leukemia (CML) patient samples, combined with bioinformatics analyses, we further examine the heterogeneity of a previously characterized in vitro imatinib-selected CD34-CD38- CML LSC population. We utilized a series of functional analyses, including single-cell metabolomic and Seahorse analyses, to validate the existence of the deepest quiescent leukemia initiators (LI) subset. Current study revealed heterogeneity of therapy resistant LSC in CML patients and their existence of two functionally distinct states. The most deeply quiescent LI suppress the expression of MC-1, yet are highly dependent on fatty acid oxidation (FAO) for their metabolic requirements and ATAC-seq demonstrated increased chromatin accessibility in this population, all consistent with an extremely primitive, quiescent stemness transcriptional signature. Importantly, the specific CREB binding protein (CBP)/β-catenin antagonist ICG-001 initiates the differentiation of LSC, including LI, decreases chromatin accessibility with differentiation and increasing expression of MC-1, CD34, CD38 and BCR-ABL1, thereby resensitizing them to imatinib. We investigated the biological aspects related to LSC heterogeneity in CML patients and demonstrated the ability of specific small molecule CBP/β-catenin antagonists to safely eliminate deeply quiescent therapy resistant CSC. These observations may represent an attractive generalizable therapeutic strategy that could help develop better protocols to eradicate the quiescent LSC population.

CD69 Marks Leukemic Regenerating Cells and Regulates Their Metabolism in AML

The treatment of relapsed or refractory (R/R) acute myeloid leukemia (AML) has been a challenge for several decades. Despite the widespread use of cytarabine-based chemotherapy and novel targeted agents, the 5-year overall survival (OS) of AML is still only about 30%, and patients mostly die from relapsed AML. Single-cell RNA sequencing (scRNA-seq) has been applied in the study of recurrence and metastasis of solid tumors, but there is still a lack of in-depth exploration of AML. Here, we investigated the transcriptome features of leukemia stem cells (LSCs) in R/R AML, explored their markers and mechanisms of relapse and chemoresistance, and provided potential therapeutic targets for the clinic. To explore the features of LSCs in R/R AML, we collected 13 bone marrow samples for scRNA-seq, including 8 samples from 4 AML patients at the time of initial diagnosis and their relapse after chemotherapy between 2017 and 2021 (median time to relapse was 333.5 days), 2 samples at initial diagnosis from AML patients with relapse-free survival for three years after chemotherapy, and 3 healthy donors (Figure.1A). Through clustering algorithm, activity analysis of relugons and copy number variation analysis comparing with normal bone marrow samples, we isolated AML cells from normal cells (Figure.1B). Changes of cell number of each cluster and leukemia stemness gene scores were used to define stemness-deficient leukemia cells (Bulk), chemotherapy-sensitive LSCs (S-LSC), and resistant LSCs (R-LSC) (Figure.1C-D). Compared with S-LSCs or Bulk, R-LSCs significantly upregulated the oxidative phosphorylation pathway (Figure.1E). Besides, we found that CD69 was significantly upgraded in R-LSCs, indicating that CD69 may represent a group of leukemic-regenerating cells (LRCs) associated with both leukemic stemness and chemoresistance (Figure.1F) . To investigate the impact of upregulated genes in R-LSC on the clinical prognosis of AML, we explored GEO database and found that the highly expressed genes in R-LSCs including CD69 were associated with a worse clinical prognosis (Figure.1G). TCGA database was used as a validation, and consistent results were obtained (Figure.1H). To explore the stemness, chemoresistance, and metabolic function of CD69+ LRCs, we sorted CD69- and CD69+ AML cells from patients. Through in vitro chemotherapy, we found that CD69+ AML cells were more resistant to chemotherapy than CD69- AML cells (Figure.2A). By clone formation assay, we found that CD69+ AML had better clonal proliferation ability (Figure.2B). To explore the stemness of CD69+ AML cells, we performed limiting dilution analysis (LDA) in vivo and found that the LSCs ratio of CD69+ AML was 109 times that of CD69- AML (p < 0.0001) (Figure.2C). Next, we explored the metabolic function of CD69+ AML cells and found that CD69+ AML cells had higher levels of pyruvate and ATP metabolism, while lactate metabolism levels were lower than those of CD69- AML cells (Figure.2D-F). After culturing with the ATP synthase inhibitor, the chemosensitivity of CD69+ AML cells was significantly improved (Figure.2G). The above evidence proves that CD69 marks a population of LRCs with high oxidative phosphorylation, stemness and chemoresistance in AML, which is consistent with the scRNA-seq results. Based on the above conclusions, we further explored the mechanism by which CD69+ LRCs maintain chemoresistance properties. By scRNA-seq analysis, we found that CD69+ LRCs had enhanced activity of STAT5 in the regulatory network (Figure.3A). We found the expression of phospho-STAT5A, MYC and BCL-2 proteins in CD69+ AML cells of patients was significantly higher than that in CD69- AML cells. To explore whether the function of CD69+ LRCs is affected by the JAK3-STAT5 pathway, we knocked down STAT5 in human AML cells. The protein expressions of MYC and BCL-2 were significantly reduced (Figure.3B). The chemosensitivity in vitro was significantly reduced in STAT5 knockdown (KD) (Figure.3C). To explore the effect of STAT5 KD on the metabolism of CD69+ LRCs, we detected metabolic index. We found that CD69+ LRCs with STAT5 KD produced decreased levels of pyruvate (Figure.3D). In conclusion, CD69 markers LRCs with high oxidative phosphorylation. CD69+ LRCs affect their metabolism and stemness through JAK3-STAT5 activation of downstream MYC and BCL-2. CD69-targeted and STAT5-targeted inhibitors may be candidate strategies for clinical R/R AML treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Splenic red pulp macrophages provide a niche for CML stem cells and induce therapy resistance

Disease progression and relapse of chronic myeloid leukemia (CML) are caused by therapy resistant leukemia stem cells (LSCs), and cure relies on their eradication. The microenvironment in the bone marrow (BM) is known to contribute to LSC maintenance and resistance. Although leukemic infiltration of the spleen is a hallmark of CML, it is unknown whether spleen cells form a niche that maintains LSCs. Here, we demonstrate that LSCs preferentially accumulate in the spleen and contribute to disease progression. Spleen LSCs were located in the red pulp close to red pulp macrophages (RPM) in CML patients and in a murine CML model. Pharmacologic and genetic depletion of RPM reduced LSCs and decreased their cell cycling activity in the spleen. Gene expression analysis revealed enriched stemness and decreased myeloid lineage differentiation in spleen leukemic stem and progenitor cells (LSPCs). These results demonstrate that splenic RPM form a niche that maintains CML LSCs in a quiescent state, resulting in disease progression and resistance to therapy.

BRD4 degradation blocks expression of MYC and multiple forms of stem cell resistance in Ph+ chronic myeloid leukemia.

In most patients with chronic myeloid leukemia (CML) clonal cells can be kept under control by BCR::ABL1 tyrosine kinase inhibitors (TKI). However, overt resistance or intolerance against these TKI may occur. We identified the epigenetic reader BRD4 and its downstream‐effector MYC as growth regulators and therapeutic targets in CML cells. BRD4 and MYC were found to be expressed in primary CML cells, CD34+/CD38− leukemic stem cells (LSC), and in the CML cell lines KU812, K562, KCL22, and KCL22T315I. The BRD4‐targeting drug JQ1 was found to suppress proliferation in KU812 cells and primary leukemic cells in the majority of patients with chronic phase CML. In the blast phase of CML, JQ1 was less effective. However, the BRD4 degrader dBET6 was found to block proliferation and/or survival of primary CML cells in all patients tested, including blast phase CML and CML cells exhibiting the T315I variant of BCR::ABL1. Moreover, dBET6 was found to block MYC expression and to synergize with BCR::ABL1 TKI in inhibiting the proliferation in the JQ1‐resistant cell line K562. Furthermore, BRD4 degradation was found to overcome osteoblast‐induced TKI resistance of CML LSC in a co‐culture system and to block interferon‐gamma‐induced upregulation of the checkpoint antigen PD‐L1 in LSC. Finally, dBET6 was found to suppress the in vitro survival of CML LSC and their engraftment in NSG mice. Together, targeting of BRD4 and MYC through BET degradation sensitizes CML cells against BCR::ABL1 TKI and is a potent approach to overcome multiple forms of drug resistance in CML LSC.

Contribution of BCR-ABL molecular variants and leukemic stem cells in response and resistance to tyrosine kinase inhibitors: a review

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm generated by reciprocal chromosomal translocation, t (9; 22) (q34; q11) in the transformed hematopoietic stem cell. Tyrosine kinase inhibitors (TKIs) target the mature proliferating BCR-ABL cells, the major CML driver, and increase overall and disease-free survival. However, mutant clones, pre-existing or due to therapy, develop resistance against TKIs. BCR-ABL1 oncoprotein activates various molecular pathways including the RAS/RAF/MEK/ERK pathway, JAK2/STAT pathway, and PI3K/AKT/mTOR pathway. Stimulation of these pathways in TKI resistant CML patients, make them a new target. Moreover, a small proportion of CML cells, leukemic stem cells (LSCs), persist during the TKI therapy and sustain the disease in the patient. Engraftment of LSCs in the bone marrow niche and dysregulation of miRNA participate greatly in the TKI resistance. Current efforts are needed for determining the reason behind TKI resistance, identification, and elimination of CML LSC might be of great need for cancer cure.

Contribution of BCR-ABL molecular variants and leukemic stem cells in response and resistance to tyrosine kinase inhibitors: a review.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm generated by reciprocal chromosomal translocation, t (9; 22) (q34; q11) in the transformed hematopoietic stem cell. Tyrosine kinase inhibitors (TKIs) target the mature proliferating BCR-ABL cells, the major CML driver, and increase overall and disease-free survival. However, mutant clones, pre-existing or due to therapy, develop resistance against TKIs. BCR-ABL1 oncoprotein activates various molecular pathways including the RAS/RAF/MEK/ERK pathway, JAK2/STAT pathway, and PI3K/AKT/mTOR pathway. Stimulation of these pathways in TKI resistant CML patients, make them a new target. Moreover, a small proportion of CML cells, leukemic stem cells (LSCs), persist during the TKI therapy and sustain the disease in the patient. Engraftment of LSCs in the bone marrow niche and dysregulation of miRNA participate greatly in the TKI resistance. Current efforts are needed for determining the reason behind TKI resistance, identification, and elimination of CML LSC might be of great need for cancer cure.

Potential Approaches Versus Approved or Developing Chronic Myeloid Leukemia Therapy.

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, continued use of these inhibitors has contributed to the increase in clinical resistance and the persistence of resistant leukemic stem cells (LSCs). So, there is an urgent need to introduce additional targeted and selective therapies to eradicate quiescent LSCs, and to avoid the relapse and disease progression. Here, we focused on emerging BCR-ABL targeted and non-BCR-ABL targeted drugs employed in clinical trials and on alternative CML treatments, including antioxidants, oncolytic virus, engineered exosomes, and natural products obtained from marine organisms that could pave the way for new therapeutic approaches for CML patients.

Shedding Light on Targeting Chronic Myeloid Leukemia Stem Cells.

Chronic myeloid leukemia stem cells (CML LSCs) are a rare and quiescent population that are resistant to tyrosine kinase inhibitors (TKI). When TKI therapy is discontinued in CML patients in deep, sustained and apparently stable molecular remission, these cells in approximately half of the cases restart to grow, resuming the leukemic process. The elimination of these TKI resistant leukemic stem cells is therefore an essential step in increasing the percentage of those patients who can reach a successful long-term treatment free remission (TFR). The understanding of the biology of the LSCs and the identification of the differences, phenotypic and/or metabolic, that could eventually allow them to be distinguished from the normal hematopoietic stem cells (HSCs) are therefore important steps in designing strategies to target LSCs in a rather selective way, sparing the normal counterparts.

Deciphering the Mechanisms of Osteoblast-Induced Resistance of Leukemic Stem Cell (LSC) in Ph+ CML: Role of PI3-Kinase, BRD4 and MYC and Development of Strategies to Overcome Osteoblast-Induced Resistance

Chronic myeloid leukemia (CML) is a stem cell neoplasms characterized by the chromosome translocation t(9;22) and the related BCR-ABL1 fusion gene. Therapy with BCR-ABL1 kinase inhibitors is highly effective in the treatment of CML and deep molecular responses are achieved in most patients. However, not all patients respond to these drugs due to resistance of leukemic stem cells (LSC). Recent data suggest that the disease-related microenvironment, known as the stem cell niche contributes to drug resistance and relapse in CML. So far, little is known about the resistance mechanisms protecting niche cells in the bone marrow of patients with CML. We have recently shown that osteoblasts are a major site of LSC-mediated resistance against BCR-ABL1-targeting drugs in CML. In the current study, we screened for drugs that are able to suppress growth and viability of osteoblasts and/or other niche-related cells and can thereby overcome drug resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50: 0.05 µM; copanlisib IC 50: 0.05 µM), the osteoblastic cell line CAL-72 (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 1 µM), primary human umbilical vein endothelial cells (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 0.5 µM) and the endothelial cell line HMEC-1 (BEZ235 IC 50: 1 µM; copanlisib IC 50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. As determined by flow cytometry, BEZ235 and copanlisib suppressed the expression of phosphorylated (p) pAKT and pS6 in endothelial cells and osteoblasts whereas rapamycin downregulated pS6 expression but did not decrease expression of pAKT. Moreover, we found that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing growth and viability of osteoblasts and endothelial cells. Furthermore, BEZ235 and copanlisib were found to overcome osteoblast-induced resistance of K562, KU812 cells, and primary CD34 +/CD38 − CML LSC against nilotinib and ponatinib. This effect was also seen when CAL-72 cells were first exposed to BEZ235 or copanlisib and washed before co-cultures with CML cells and BCR-ABL1 inhibitors were prepared, suggesting that osteoblast inhibition was a crucial event capable of disrupting LSC resistance in these co-cultures. Of all other drugs tested, only the BRD4-targeting drug JQ1 was found to suppress CAL72-induced resistance in the CML cell lines KU812 and K562, suggesting that osteoblast-induced resistance of CML cells is also mediated by a BRD4-MYC pathway. In a next step, we examined the expression of resistance-mediating immune checkpoint molecules on CML cells (KU812, K562, LSC) and on osteoblasts by flow cytometry. We found that CML cells and CAL72 cells constitutively express PD-L1 and that interferon-gamma (IFN-G) promotes the expression of PD-L1 in all cell types tested. Moreover, we found that the BRD4 blocker JQ1 and the BRD4-degrader dBET6 suppress the IFN-G-induced upregulation of PD-L1 in CML LSC and osteoblasts. In conclusion, our data show that osteoblast-induced resistance of CML stem cells is mediated by a PI3K-dependent pathway and BRD4/MYC, and that BRD4-inhibition or BRD4-degradation counteracts osteoblast-induced resistance of CML (stem) cells against BCR-ABL1 inhibitors and PD-L1 expression on CML LSC and osteoblasts. We hypothesize that checkpoint inhibition may assist in drug-induced eradication of CML LSC and thus in the development of curative drug therapies in Ph + CML. DisclosuresHoermann: Novartis: Honoraria. Gleixner: Pfizer: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

Inhibition of Folate Metabolism Drives Autophagy-Dependent Differentiation and Reduces Survival of Therapy-Resistant Leukaemic Stem Cells

Metabolic rewiring is an important hallmark of cancer. The folate metabolism pathway, also known as one-carbon (1C) metabolism, allows for transfer of 1C units through folate intermediates for biosynthetic processes, including precursors for DNA synthesis. Recent studies have shown that enzymes involved in the mitochondrial arm of 1C metabolism are overexpressed in a subset of aggressive cancers and that their expression affects responses to anti-metabolite drug treatments. However, the role of 1C metabolism in therapy resistant leukemic stem cells (LSCs) is currently unknown. Therefore, we aimed to investigate the activity and the impact of genetic and pharmacological inhibition of folate enzymes in primitive chronic myeloid leukaemia (CML) cells.We initially performed transcriptomic analysis of CD34+38- cells, from individuals with chronic phase CML (E-MTAB-2581). This revealed a significant upregulation of folate metabolism genes in CML LSCs, including serine hydroxymethyltransferase (SHMT2; p≤0.05), a key mitochondrial enzyme. To assess the activity of 1C metabolism in primitive cells we performed gas chromatography-mass spectrometry-mediated secretomic analysis using patient-derived, c-KIT enriched CML cells, which revealed a significant increase in the exchange rate of formate (folate intermediate necessary for purine synthesis) in CML cells, when compared to the secretome of normal counterparts (p<0.05). This reinforced the idea that 1C metabolism may be a metabolic dependency in CML.Following CRISPR-Cas9-mediated SHMT2 knockout (KO) in CML cell line, we observed a significant decrease in growth rate, together with a decrease in glycolytic capacity and oxygen consumption rate (p<0.01), suggesting impairment in proliferation and central carbon metabolism. Further metabolic characterisation of CML SHMT2 KO cells using liquid chromatography-mass spectrometry demonstrated a significant increase in AICAR, a purine biosynthesis intermediate and an AMP activated kinase (AMPK) activator. This prompted us to investigate the effect of 1C metabolism inhibition on AMPK. We found that AMPK phosphorylation on the conserved Thr 172 (a site that is phosphorylated under energy stress) was increased in SHMT2 KO cells, with similar effect seen following pharmacological inhibition of both SHMT2 and its cytosolic counterpart SHMT1 using SHIN1,which also promoted AMPK-dependent phosphorylation of the autophagy-inducing kinase ULK1 and downstream ULK1 target ATG13. Moreover, analysis of mitochondrial fraction revealed accumulation of mitochondrial fission related protein DRP1 and the mitophagy receptor NIX on mitochondria, hinting towards cellular interplay between 1C metabolism and mitochondrial homeostasis.Phenotypically, both pharmacological and genetic inhibition of SHMT1/2 induced the expression of erythropoiesis markers CD71 and Glycophorin A, which was reversed following formate supplementation. CRISPR-Cas9-mediated double AMPKα1/α2 KO revealed that the increased expression of these erythropoiesis markers following SHMT1/2 inhibition was independent of AMPK activity. Conversely, while NIX KO had no effect, pharmacological inhibition of ULK1 kinase activity, or genetic inhibition of ULK1 and ATG7 (protein important for autophagosome formation), prevented increased expression of CD71/Glycophorin A following SHMT1/2 inhibition.We next investigated the effect of 1C metabolism inhibition on differentiation and survival of primary CML cells. Of clinical relevance, pharmacological inhibition of SHMT1/2 promoted erythroid maturation of CD34+ CML cells (measured by expression of CD71, CD44, CD36 and Glycophorin A) when challenged with erythropoietin, which sensitises primitive cells to erythroid lineage commitment. Lastly, pharmacological inhibition of 1C metabolism decreased the colony formation capacity of CD34+ CML by 50%, with minimum effect on normal CD34+ cells. Moreover, combination treatment of SHIN1 with imatinib, a frontline treatment for CML patients, further increased the sensitivity of primary CML cells to imatinib by 40%.Overall, our novel findings indicate that disruption of the folate metabolism pathway inhibits central carbon metabolism in CML cells, promotes autophagy dependent, but AMPK independent maturation phenotype and has detrimental effect on the survival of primitive CML cells. DisclosuresNo relevant conflicts of interest to declare.

Analyzing normal and disrupted leukemic stem cell adhesion to bone marrow stromal cells by single-molecule tracking nanoscopy.

Leukemic stem cells (LSCs) adhere to bone niches through adhesion molecules. These interactions, which are deeply reorganized in tumors, contribute to LSC resistance to chemotherapy and leukemia relapse. However, LSC adhesion mechanisms and potential therapeutic disruption using blocking antibodies remain largely unknown. Junctional adhesion molecule C (JAM-C, also known as JAM3) overexpression by LSCs correlates with increased leukemia severity, and thus constitutes a putative therapeutic target. Here, we took advantage of the ability of nanoscopy to detect single molecules with nanometric accuracy to characterize junctional adhesion molecule (JAM) dynamics at leuko-stromal contacts. Videonanoscopy trajectories were reconstructed using our dedicated multi-target tracing algorithm, pipelined with dual-color analyses (MTT2col). JAM-C expressed by LSCs engaged in transient interactions with JAM-B (also known as JAM2) expressed by stromal cells. JAM recruitment and colocalization at cell contacts were proportional to JAM-C level and reduced by a blocking anti-JAM-C antibody. MTT2col revealed, at single-molecule resolution, the ability of blocking antibodies to destabilize LSC binding to their niches, opening opportunities for disrupting LSC resistance mechanisms.

Splenic Red Pulp Macrophages Provide a Secondary Stem Cell Niche for Chronic Myeloid Leukemia Stem Cells

Leukemic stem cells (LSCs) are responsible for initiation, progression and relapse of the disease. Chronic myeloid leukemia (CML) is a classical stem-cell driven malignancy and represents an attractive model to study LSCs. Resistance of LSCs to treatment is mediated by cell-intrinsic characteristics but also by the interactions of LSCs with their microenvironment. LSCs depend on signals from their surrounding niche in the bone marrow (BM) and spleen in order to maintain stem cell characteristics such as quiescence and self-renewal. The BM as a niche for LSCs is well-investigated and several therapeutic targets are under consideration.Splenomegaly is a hallmark of CML. Nevertheless, the role of the spleen on leukemia and LSCs in unknown. Therefore, this project aims to investigate the potential role of the spleen as an independent secondary niche for CML stem cells and its contribution to disease development and progression.In order to study the role of the spleen in CML development and progression, we retrovirally transduced FACS-purified Lineage- Sca-1+ c-Kit+ BM cells (LSKs) with pMSCV-p210BCR/ABL-IRES-GFP and injected transduced LSKs into splenectomized or sham operated mice. Splenectomy prior to CML induction significantly improved survival compared to sham operated controls (median survival 31 vs. 22 days; p=0.0006) with 20 % of splenectomized animals surviving longer than 90 days (endpoint). In addition, splenectomized animals showed a 3.7-fold reduction (p=0.002) in LSC numbers in the BM when analyzed 18 days after CML induction, suggesting that the spleen influences the disease burden in the BM.We next analyzed the leukemic compartment in the BM and spleen and found a significant enrichment of leukemic stem and progenitor cells in the spleen (20 % spleen vs. 10 % BM; % leukemic stem and progenitor cells of total leukemic cells; p=0.01; 19-fold increase in total amount of LSCs in the spleen; p =0.007). To functionally confirm the phenotypic increase in splenic LSCs, we performed secondary transplantations of total leukemic cells from the BM or spleen at limiting dilution into lethally irradiated secondary recipients. The analysis revealed a significantly higher frequency of LSCs in the spleen compared to the BM (1/41'703 vs. 1/432'594; p=0.02).In addition, we determined the localization of LSCs in the spleen by confocal microscopy on optically cleared spleens. Splenic LSCs were exclusively localized within the red pulp and in close proximity to macrophages. It was previously shown that hematopoietic stem cells reside in direct contact with macrophages in the red pulp of the spleen during extramedullary hematopoiesis (Dutta et al., JEM, 2015). Therefore, we depleted macrophages in CML mice using clodronate liposomes. Spleen weight (867mg vs. 249mg; p<0.0001) and numbers of splenic LSCs (23-fold; p=0.001) were significantly reduced in clodronate treated animals. Importantly, clodronate treatment did not affect LSC viability, as indicated by comparable amounts of AnnexinV+ cells in both groups.In summary, we show that the interaction of splenic niche cells (red pulp macrophages) with LSCs results in the generation of a big reservoir of LSCs which contribute to CML progression. Therefore, our study strongly suggest the presence of an independent secondary splenic LSC niche in addition to the well characterized BM LSC niche in CML. DisclosuresNo relevant conflicts of interest to declare.