Immature Progenitors Research Articles

PDX1 as a Marker of Early Differentiation of Human Pancreatic Duct Cells.

Epithelial progenitor cells of the primitive pancreatic ducts differentiate in 3 directions, which is accompanied by changes in cell phenotype. We analyze the distribution of the pancreatic and duodenal homeobox 1 (PDX1) in the epithelial cells of pancreatic ducts during the prenatal development in order to determine the potential use of this marker as an indicator of pancreatic cell maturation. Pancreatic autopsies from human fetuses (gestation weeks 17-23) were examined by immunohistochemical methods. In general, PDX1 was detected in the epithelial cells that are involved in various morphogenetic processes related to the growth and branching of the fetal pancreatic ducts. The intensive positive reaction to PDX1/cytokeratin 19 indicates immature (progenitor) polypotent ductal cells.

ATP citrate lyase is an essential player in the metabolic rewiring induced by PTEN loss during T-ALL development.

Alterations inactivating the tumor suppressor gene PTEN drive the development of solid and hematological cancers, such as T-cell acute lymphoblastic leukemia (T-ALL), whereby PTEN loss defines poor-prognosis patients. We investigated the metabolic rewiring induced by PTEN loss in T-ALL, aiming at identifying novel metabolic vulnerabilities. We showed that the enzyme ATP citrate lyase (ACLY) is strictly required for the transformation of thymic immature progenitors and for the growth of human T-ALL, which remain dependent on ACLY activity even upon transformation. Whereas PTEN mutant mice all died within 17 weeks, the concomitant ACLY deletion prevented disease initiation in 70% of the animals. In these animals, ACLY promoted BCL-2 epigenetic upregulation and prevented the apoptosis of pre-malignant DP thymocytes. Transcriptomic and metabolic analysis of primary T-ALL cells next translated our findings to the human pathology, showing that PTEN-altered T-ALL cells activate ACLY and are sensitive to its genetic targeting. ACLY activation thus represents a metabolic vulnerability with therapeutic potential for high-risk T-ALL patients.

Understanding MDS stem cells: Advances and limitations

In work spanning several decades, extensive studies have focused on the properties of malignant stem cells that drive the pathogenesis of acute myeloid leukemia (AML). However, relatively little attention has been devoted to several serious myeloid malignancies that occur prior to the onset of frank leukemia, including myelodysplastic syndrome (MDS). Like leukemia, MDS is hypothesized to arise from a pool of immature malignant stem and progenitor cells (MDS-SCs) that serve as a reservoir for disease evolution and progression1. While multiple studies have sought to identify and characterize the biology and vulnerabilities of MDS-SCs, yet translation of scientific concepts to therapeutically impactful regimens has been limited. Here, we evaluate the currently known properties of MDS-SCs as well as the post-transcriptional mechanisms that drive MDS pathogenesis at a stem and progenitor level. We highlight limits and gaps in our characterization and understanding of MDS-SCs and address the extent to which the properties of MDS-SC are (and can be) inferred from the characterization of LSCs.

Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition.

Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action.

Lenalidomide and pomalidomide modulate hematopoietic cell expansion and differentiation in the presence of MSC

Cytopenia is a well-documented complication in the treatment of hematological malignancies with lenalidomide and pomalidomide. Although prior studies have highlighted direct effects on hematopoietic cells to explain this adverse effect, the involvement of hematopoietic-supportive stroma remains less understood. This study examined the effects of lenalidomide/pomalidomide on the expansion and differentiation of human CD34+ hematopoietic stem/progenitor cells (HSPCs) in vitro, in co-culture with human bone-marrow mesenchymal stromal/stem cells (MSCs). Our findings indicate that lenalidomide/pomalidomide increases the population of immature CD34+CD38− cells while decreasing the number of mature CD34+CD38+ cells, suggesting a mechanism that inhibits early HSPC maturation. This effect persisted across myeloid, megakaryocytic, and erythroid lineages, with MSCs playing a key role in preserving immature progenitors and inhibiting their differentiation. Furthermore, in myeloid differentiation assays augmented by granulocyte-colony stimulating factor, lenalidomide/pomalidomide not only enhanced the presence of CD34+ cells with mature myeloid markers such as CD11b but also reduced the populations lacking CD34 yet positive for these markers, irrespective of MSC presence. Thus, while MSCs support the presence of these immature cell populations, they simultaneously inhibit their maturation. This finding provides novel mechanistic insights into lenalidomide- and pomalidomide-induced cytopenia, and could guide therapeutic strategies for its mitigation.

Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting.

Haematopoiesis dysregulation with the presence of immature myeloid and erythroid immunosuppressive cells are key characteristics of the immune escape phase of tumour development. Here, the role of in vitro generated B16F10 tumour cell-derived extracellular vesicles (tEVs) as indirect cellular communicators, participating in tumour-induced dysregulation of haematopoiesis, was explored. The isolated tEVs displayed features of small EVs with a size range of 100-200nm, expressed the common EV markers CD63, CD9, and Alix, and had a spherical shape with a lipid bilayer membrane. Proteomic profiling revealed significant levels of angiogenic factors, particularly vascular endothelial growth factor (VEGF), osteopontin, and tissue factor, associated with the tEVs. Systemic administration of these tEVs in syngeneic mice induced splenomegaly and disrupted haematopoiesis, leading to extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and the development of anaemia. These effects closely mirrored those observed in tumour-bearing mice and were not seen after heat inactivating the tEVs. In vitro studies demonstrated that tEVs independently induced the expansion of bone marrow granulocytic myeloid suppressor cells and B cells while reducing the frequency of cells in the erythropoietic lineage. These effects of tEVs were significantly abrogated by the blockade of VEGF or heat inactivation. Our findings underscore the important role of tEVs in dysregulating haematopoiesis during the immune escape phase of cancer immunoediting, suggesting their potential as targets for addressing immune evasion and reinstating normal hematopoietic processes.

Memory loss and aberrant neurogenesis in mice exposed to patient anti-N-methyl-d-aspartate receptor antibodies

ObjectiveAnti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis results in chronic epilepsy and permanent cognitive impairment. One of the possible causes of cognitive impairment in anti-NMDAR could be aberrant neurogenesis, an established contributor to memory loss in idiopathic drug-resistant epilepsy. We developed a mouse model of anti-NMDAR encephalitis and showed that mice exposed to patient anti-NMDAR antibodies for 2 weeks developed seizures and memory loss. In the present study, we assessed the delayed effects of patient-derived antibodies on cognitive phenotype and examined the corresponding changes in hippocampal neurogenesis. MethodsMonoclonal anti-NMDAR antibodies or control antibodies were continuously infused into the lateral ventricle of male C56BL/6J mice (8–12 weeks) via osmotic minipumps for 2 weeks. The motor and anxiety phenotypes were assessed using the open field paradigm, and hippocampal memory and learning were assessed using the object location, Y maze, and Barnes maze paradigms during weeks 1 and 3–4 of antibody washout. The numbers of newly matured granule neurons (Prox-1+) and immature progenitor cells (DCX+) as well as their spatial distribution within the hippocampus were assessed at these time points. Bromodeoxyuridine (BrdU, 50 mg/kg, i.p., daily) was injected on days 2–12 of the infusion, and proliferating cell immunoreactivity was compared in antibody-treated mice and control mice during week 4 of the washout. ResultsMice infused with anti-NMDAR antibodies demonstrated spatial memory impairment during week 1 of antibody washout (p = 0.02, t-test; n = 9–11). Histological analysis of hippocampal sections from these mice revealed an increased ectopic displacement of Prox-1+ cells in the dentate hilus compared to the control-antibody-treated mice (p = 0.01; t-test). Mice exposed to anti-NMDAR antibodies also had an impairment of spatial memory and learning during weeks 3–4 of antibody washout (object location: p = 0.009; t-test; Y maze: p = 0.006, t-test; Barnes maze: p = 0.008, ANOVA; n = 8–10). These mice showed increased ratios of the low proliferating (bright) to fast proliferating (faint) BrdU+ cell counts and decreased number of DCX+ cells in the hippocampal dentate gyrus (p = 0.006 and p = 0.04, respectively; t-tests) suggesting ectopic migration and delayed cell proliferation. SignificanceThese findings suggest that memory and learning impairments induced by patient anti-NMDAR antibodies are sustained upon removal of antibodies and are accompanied by aberrant hippocampal neurogenesis. Interventions directed at the manipulation of neuronal plasticity in patients with encephalitis and cognitive loss may be protective and therapeutically relevant.

B-NHL Cases in a Tertiary Pediatric Hematology-Oncology Department: A 20-Year Retrospective Cohort Study.

Non-Hodgkin lymphoma (NHL) is among the five most common pediatric cancer diagnoses in children and adolescents and consists of a heterogeneous group of lymphoid tissue malignancies -with B-cell-derived NHL accounting for nearly 80% of cases. Novel and high-throughput diagnostic tools have significantly increased our understanding of B-NHL biology and molecular pathogenesis, leading to new NHL classifications and treatment options. This retrospective cohort study investigated 17 cases of both mature B-cell NHL (Burkitt lymphoma or BL; Diffuse large B-cell lymphoma or DLBCL; Primary mediastinal large B-cell lymphoma or PMBCL; Follicular lymphoma or FL) and immature B-cell progenitor NHL (B-lymphoblastic lymphoma or BLL) that were treated in a tertiary Pediatric Hematology-Oncology Department during the last 20 years. Modern NHL protocols for children, adolescents, and young adults, along with the addition of rituximab, are safe and efficient (100% overall survival; one relapse). Elevated ESR was more prevalent than elevated LDH. Analyses have focused on immune reconstitution (grade ≥3 infections, lymphocyte and immunoglobulin levels recovery) and body-mass-index changes post-treatment, late effects (in 53% of patients), and the presence of histology markers BCL2, BCL6, CD30, cMYC, and Ki-67%. One patient was diagnosed with a second malignant neoplasm (papillary thyroid cancer).

Identification of a myofibroblast differentiation program during neonatal lung development.

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFBs) and a stable but poorly described population of lipid-rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFBs). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single-cell RNA sequencing and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a MyoFB differentiation program that is distinct from other mesenchymal cell types and increases the known repertoire of mesenchymal cell types in the neonatal lung.

Clinical features of immature leukemias in children.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), mixed phenotypic acute leukemia (MPAL), and acute myeloid leukemia with minimal differentiation (AML-M0) all originate from immature hematopoietic progenitor cells and have a poor prognosis. We investigated the clinical characteristics of these immature leukemias in 17 children (ETP-ALL: 8, MPAL: 5, AML-M0: 4) at seven institutions. Clinical and laboratory findings were comparable across disease types. Eleven and six patients received ALL- and AML-oriented induction chemotherapy, with six and four achieving complete remission (CR), respectively. Five additional patients achieved CR after salvage with the other type of chemotherapy. Eight patients received hematopoietic cell transplantation (HCT) in first CR, and six survived without relapse. However, six of seven patients who did not receive HCT during first CR relapsed; all underwent HCT later, and only three survived. The 5-year event-free survival (EFS) and overall survival (OS) rate were 37% and 69%, respectively. Patients who achieved CR after induction chemotherapy and received HCT in first CR had favorable EFS and OS. Notably, all patients who received HCT in first CR survived 5years after diagnosis. Appropriate induction chemotherapy and HCT in first CR could improve the outcome of immature leukemias.

Cancer Stem Cells (CSCs) and its various biomarkers for treatment of human cancers: Review article

Traditional cancer treatments often fail to target specific cells, leading to therapy resistance and recurrence. Stem cell biology offers new potential for cancer treatment, including self-renewal, migration, differentiation, and modulatory effects. This review discusses stem cell use in cancer treatment, clinical applications, risks, and future directions for improving cancer outcomes. The kind of treatment chosen is determined by the kind, stage, and goal of the cancer. The first line of treatment for the direct removal of solid tumors that are localized is surgery. Radiotherapy damages the DNA of cancer cells, which can kill tumors. Chemotherapy helps slow down or stop tumour growth by using extremely toxic drugs. Many stem cell-based approaches are currently being studied in preclinical trials, and they present both exciting opportunities and difficult challenges for the treatment of cancer. Epigenetic mutations in normal stem cells or in precursor/progenitor cells give rise to CSCs, also known as stem-like cells, immature progenitors of tumor cells, or tumor-initiating cells. Tumor tissues contain CSCs, which are crucial for the development, spread, and recurrence of cancer. Thus, targeting CSCs may hold the key to treating different kinds of solid tumors. This review discusses the biological roles of stem cells and their diverse applications in cancer treatment. HSC transplantation is effective for hematologic cancers like leukaemia, multiple myeloma, and lymphomas. Immunomodulatory MSCs have shown high effectiveness in reducing GVHD cases and repairing injured tissues. MSCs and NSCs have also been investigated for tumour-tropic properties.

Abstract 4620: Exploring the role of GHRH antagonist MIA-602 in overcoming doxorubicin resistance in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is characterized by the uncontrolled proliferation of immature hematopoietic progenitors, often associated with a poor prognosis. Conventional therapies involve chemotherapy and bone marrow stem cell transplantation. The growth hormone releasing hormone receptor (GHRH-R) antagonist MIA-602 has exhibited inhibitory effects on the growth of various human cancer cell lines, including AML cells. This study aimed to investigate the impact of MIA-602 alone and in combination with doxorubicin on three doxorubicin-resistant AML cell lines, K-562, U-937, and KG-1A. Initial examinations confirmed the presence of GHRH receptors in both wild-type and doxorubicin-resistant cell lines through western blot analysis. Doxorubicin was administered at concentrations of 0.005, 0.01, and 0.05 μg/ml, while MIA-602 was used at 5 μmol/L. Subsequent experiments involved culturing both wild-type and doxorubicin-resistant clones with MIA-602 at concentrations ranging from 0.05 μmol/L to 5 μmol/L for 24 and 48 hours. The results revealed a significant, dose- and time-dependent reduction in cell proliferation across all six cell lines. Both wild-type and doxorubicin-resistant clones exhibited a comparable decrease in cell proliferation when exposed to MIA-602 at concentrations greater than 0.05 μmol/L (p < 0.05) after 24 and 48 hours. In conclusion, our study demonstrates that these three AML cell lines and their doxorubicin-resistant clones remain susceptible to GHRH antagonist MIA-602. These findings may pave the way for the development of novel therapies or drug combinations using GHRH antagonists such as MIA-602 for treating AML. This is of particular interest for those who are resistant to conventional chemotherapy, further broadening the spectrum of treatment options available. Additional investigations are warranted to progress these findings to in vivo xenograft models. Citation Format: Simonetta I. Gaumond, Joel Costoya, Andrew V. Schally, Joaquin J. Jimenez. Exploring the role of GHRH antagonist MIA-602 in overcoming doxorubicin resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4620.

Abstract 2852: Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells

Abstract Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system derived from migratory neural crest cells that have committed to become sympathetic neuroblasts, but their definitive differentiation is blocked. Neuroblastoma cell identity depends on the adrenergic core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive cell proliferation and the oncogenic gene expression program. Retinoic acid is used as differentiation therapy for patients with high-risk neuroblastoma, with the goal to block proliferation and drive sympathetic neuronal differentiation. We recently showed that retinoic acid re-organizes the enhancer landscape of neuroblastoma and promotes a new retino-sympathetic cell state characterized by MYCN downregulation, proliferative arrest, and sympathetic neuronal differentiation. However, the effects of retinoic acid on epigenetic rewiring of the adrenergic CRC and suppression of MYCN expression are reversible in vitro and in vivo, leading to tumor re-growth when the retinoic acid is withdrawn. Therefore, we sought to determine if retinoid-induced differentiation can be forced to progress to irreversible neuronal maturation by the addition of inhibitors of epigenetic modifying enzymes. Here, we conducted a drug screen of epigenetic modifying drugs alone and in combination with retinoic acid to determine their effects on neuroblastoma cell growth and differentiation. In this screen, we found that an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, PF-9363, synergistically inhibits neuroblastoma cell growth in combination with retinoic acid. Moreover, retinoid-resistant neuroblastoma cell lines can be sensitized to retinoic acid when combined with a KAT6 inhibitor. Importantly, this combination treatment renders the differentiated cell state irreversible, such that it is maintained after retinoic acid is withdrawn, with continued suppression of the adrenergic CRC and MYCN expression. In conclusion, the retino-sympathetic differentiated cell state induced by retinoic acid in neuroblastoma becomes irreversible when the cells are also treated with an inhibitor of the KAT6A/B histone H3K23 acetyltransferases, implicating the essential role of these enzymes in restoring the proliferative immature progenitor phenotype in adrenergic neuroblastoma cells. Citation Format: Nina Weichert-Leahey, Alla Berezovskaya, Mark Zimmerman, Brian J. Abraham, Adam W. Durbin, A Thomas Look. Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2852.

Cryopreserved cGMP-compliant human pluripotent stem cell-derived hepatic progenitors rescue mice from acute liver failure through rapid paracrine effects on liver cells

BackgroundLiver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function.MethodsHuman PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses.ResultsGStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation.ConclusionsWe generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.

Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Detection of Cancer Stem Cells in Normal and Dysplastic/Leukemic Human Blood.

The hierarchical organization of the leukemic stem cells (LSCs) is identical to that of healthy counterpart cells. It may be split into roughly three stages: a small number of pluripotent stem cells at the top, fewlineage-restricted cells in the middle, and severalterminally differentiated blood cells at the bottom. Although LSCs can differentiate into the hematopoietic lineage, they can also accumulate as immature progenitor cells, also known as blast cells. Since blast cells are uncommon in healthy bloodstreams, their presence might be a sign of cancer. For instance, a 20% blast cutoff in peripheral blood or bone marrow is formally used to distinguish acute myeloid leukemia from myelodysplastic neoplasms, whichis essential to plan the patients' management. Many techniques may be useful for blast enumeration: one of them is flow cytometry, which can perform analyses on many cells by detecting the expression of cell surface markers. Leukemic and non-leukemic blast cells might indeed be characterized by the same surface markers, but these markers are usually differently expressed. Here we propose to use CD45, in combination with CD34 andother cell surface markers, to identify and immunophenotype blast cells in patient-derived samples.

Differentiation therapy as a new multidisciplinary approach to the treatment of human brain glioma

Differentiation therapy as a new multidisciplinary approach to the treatment of human brain glioma

Targeting Sumoylation Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment

Acute myeloid leukemia (AML) is a hematopoietic cancer characterized by aberrant immature myeloid progenitor blasts in bone marrow and peripheral blood. Venetoclax (VEN), a BCL-2 inhibitor, is FDA-approved for AML treatment with hypomethylating agents (HMA). Despite the improvement of VEN and HMA, not all patients respond, and most patients develop resistance. This presents an urgent need for new therapies to overcome VEN resistance and enhance VEN efficacy. We propose SUMOylation inhibition as a novel therapy to address this need. SUMOylation regulates protein function by covalently attaching Small Ubiquitin-like MOdifier (SUMO) proteins to target proteins. SUMOylation plays an important role in tumor progression and immune response. Our study aims to evaluate the effects of SUMOylation inhibition on anti-AML activity of VEN and the mechanism. We firstly generated VEN-resistant cell lines Molm13-VR and MV411-VR by culturing Molm13 and MV4-11 cells in increasing doses of VEN. SUMO E1 (SAE2) and global SUMOylation levels were remarkably upregulated in VEN-resistant cell lines, suggesting SUMOylation plays a role in VEN resistance. We evaluated the antileukemic effects of TAK-981 (subasumstat), a novel and specific SUMO E1 inhibitor, alone and in combination with VEN, using AML cell lines and primary blasts isolated from refractory/relapsed (R/R) AML patients. Different AML cell lines and blast samples showed various sensitivities to VEN (IC 50 10nM ~ 5,000nM), but significant synergism was observed with TAK-981 and VEN in cell viability assay. TAK-981 synergized with VEN at inducing AML cell apoptosis determined by annexin-V staining with flow cytometry as well as increased levels of cleaved-PARP and cleaved caspase-3 by western blotting. The mechanism of action of VEN is dependent on BCL-2 mediated mitochondrial function and metabolism. We assessed mitochondrial membrane potential using TMRM probe. TAK-981 reduced mitochondrial membrane potential in both VEN-sensitive and resistant AML lines, further in combination with VEN. Because mitochondrial membrane potential is maintained by normal mitochondrial structure, we performed electron microscopy to determine mitochondrial structure. VEN had limited effect on mitochondria in resistant cells. However, TAK-981 induced abnormal ultrastructure mitochondria with fewer cristae, worsened by TAK-981+VEN combination. Since TAK-981 treatment caused mitochondrial structure defects, we determine the mitochondrial function using Seahorse XF assay. VEN decreased oxygen consumption rate (OCR), indicating inhibition of oxidative phosphorylation (OXPHOS), further suppressed by TAK-981. Metabolomic profiling by mass spectrometry indicated TAK-981+VEN treated AML cells displayed striking decreases in TCA cycle intermediates and reduced electron transport chain (ETC) activity. Pathway analysis of the metabolomics data revealed significant enrichment in amino acids metabolism. Notably, RNA-sequencing and GSEA analysis showed consistent findings that TAK-981+VEN showed suppressed signaling pathways in mitochondrial function, glycolysis and amino-acid metabolisms, indicating TAK-981 targeted mitochondrial metabolism to enhance VEN activity. We then determined whether TAK-981 enhanced antileukemic effect of VEN ex vivo and in vivo. TAK-981 synergized with VEN at reducing colony formation capacity in colony formation assay using leukemia stem cells (LSCs) isolated from relapse AML blasts. More importantly, TAK-981 showed remarkable therapeutic effect in patient-derived xenograft (PDX) mouse model engrafted with blasts isolated from R/R AML patients, and significantly synergized with VEN at extending mice survival in vivo. In conclusion, TAK-981 enhances the antileukemic activity of VEN by targeting mitochondria function and metabolism, offering a potent therapy for refractory/relapsed AML.

Transient Differentiation-State Plasticity during Initiation of Pediatric Acute Lymphoblastic Leukemia

Introduction Leukemia is characterized by oncogenic lesions that result in a block of differentiation while at the same time intratumor phenotypic plasticity is retained. It is unclear how these two phenomena arise during leukemogenesis in humans. Here we characterized T-cell acute lymphoblastic leukemia (T-ALL), as a model disease to investigate the coherence between leukemia initiating cells (LICs), developmental arrest, and phenotypic plasticity. Methods We characterized 31 primary T-ALL samples for differentiation state heterogeneity using multi parameter flow cytometry. Subsequently, for 5 patients, phenotypic subpopulations were single cell sorted. As leukemic cells do not expand in vitro primary template-directed amplification (PTA) was used to amplify the genome of single blasts to obtain enough DNA for WGS analysis with nucleotide resolution. Shared and unique mutations were used to construct retrospective lineage trees and assess the hierarchy of phenotypically diverse blasts. Results To assess phenotypic differentiation state heterogeneity in T-ALL, we designed a 17-parameter flow cytometry panel to discriminate T-lymphoid developmental cell populations. We characterized 31 samples (median blast count 98% (IQR = 94 - 99)), and defined immunophenotypic subpopulations as populations with a size of >10% of the total CD7+ population. Differentiation state heterogeneity (> 2 subpopulations) was observed in 26 out of the 31 T-ALL patients (83.9%). Interestingly, the most immature CD4-CD8-CD3- “DN-like” differentiation state was found in all samples, suggesting that developmental hierarchy is maintained. We then questioned whether genetic determinants were driving the phenotypic diversity we observed. Therefore 5 T-ALL patients harboring translocations of oncogenes commonly seen in T-ALL were selected, namely, TLX1 (n=1) , LMO2 (n=2) , TAL1 (n=2) and TLX3 (n=1) . For each patient cells of each differentiation state were single cell and bulk sorted for single cell WGS and bulk WGS, respectively. Structural variant analysis confirmed presence of the oncogenic translocation in all single blasts, validating their (pre-)leukemic origin. Moreover, driver analysis showed that type B mutations in e.g., NOTCH1 and PHF6 were shared among all blasts. Interestingly, there were no potential genetic drivers found that were unique to a subpopulation. Neutral passenger mutations were therefore used to construct phylogenetic trees. Herein, blasts with similar phenotypic differentiation states were genetically closer related than blasts of different phenotypes, revealing the heritability of phenotypes (Fig. 1A). The biased distribution of immunophenotypes across the different branches of the phylogenetic tree was then confirmed in bulk WGS samples of sorted phenotypic populations. Suggesting that switching between phenotypes is not a frequent process. Next, the plasticity of T-ALL was assessed by identifying the phenotypic cell state of the LIC. We assessed V(D)J recombination per single blast and identified monoclonal V(D)J recombination for most of the blasts. Moreover, in TAL1 T-ALL we observed monoclonal TRG, TRB and TRA translocation. This suggested that the LIC had undergone complete V(D)J recombination and was a more mature T-cell progenitor. However, blasts with an immature CD4-CD8- (DN) phenotype had undergone the same V(D)J recombination suggesting phenotypic de-differentiation of the LIC. Additionally, in TLX1 T-ALL, the blasts with the more mature CD4+CD8+ double positive (DP) phenotype had monoclonal TRG and TRB gene recombination, but polyclonal TRA recombination, whereas cells with a more immature DN phenotype had no TRA recombination (Fig. 1B). This indicated an immature T-cell progenitor as the LIC and continued differentiation after leukemia initiation resulting in DP cells with unique TRA recombination. Conclusion In conclusion, we show immunophenotypic differentiation state heterogeneity in diagnostic primary T-ALL samples. By coupling phenotypic data with novel single cell whole genome sequencing techniques, we identified heritability of phenotypic cell states. Moreover, we reveal the ability of the LIC to differentiate and de-differentiate after leukemia initiation. Taken together our results demonstrate a transient period of plasticity during leukemia initiation where phenotypic switches appear unidirectional.

Optimal Therapeutic Strategies in Relapsed/Refractory AML with Prior Exposure to Venetoclax Based Therapy

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy arising from immature myeloid progenitors with notoriously poor outcomes, particularly in the elderly. Venetoclax (Ven) plays an essential role in acute myeloid leukemia (AML) directed therapy as it is approved for frontline therapy in combination with azacitidine (Aza), decitabine (Dec), or low-dose cytarabine (LDAC) for patients aged 75 years and older, or those with significant comorbidities. However, venetoclax-based therapy is not curative, and the ideal salvage regimen is unclear in these patients. We performed a retrospective review that analyzed a single center's venetoclax-based regimens to identify rational subsequent lines of therapy after failing venetoclax. Methods: We conducted a retrospective review of adult patients at Roswell Park Comprehensive Cancer Center who had a confirmed AML diagnosis and received a venetoclax-based regimen as upfront therapy between January 2015-February 2023. Demographics, prior hematological disorders, ELN risk stratification, Eastern Cooperative Oncology Group (ECOG) performance status, treatment received in upfront vs relapsed/refractory settings, date of remission/relapse, disease-specific variables [molecular data, cytogenetics], and outcomes [duration of response, time to progression, MRD status, and overall survival] were collected. Of note, in patients who received upfront venetoclax regimen, the subsequent salvage therapy was grouped by targeted (Gliterinib, Enasidenib, Ivosidenib), low-intensity (single agent hypomethylating therapy, gemtuzumab, ozogamicin), or intensive groups (CLAG-M) and analysis was performed for progression-free survival (PFS). The patient demographics and clinical characteristics were summarized by group (i.e. response, regimens) using the Mann-Whitney U and Fisher's exact test. Survival functions were described by the Kaplan-Meier method and compared with the Log-rank test. Analyses were performed in SAS v9.4 (Cary, NC) at a significance level of 0.05. Results: The retrospective study analyzed 97 patients. In the upfront analysis, the median age was 73.4 years (range: 51.4-89.8) and 64 (66.0%) of the patients were male. 65 patients (67.0%) received Aza/Ven, 26 (26.8%) Dec/Ven, and 6 (6.2%) LDAC/Ven. Per ELN 2022 criteria, 13.3% of patients were stratified as favorable,12.2% as intermediate, and 74.4% as adverse risk. Of the adverse risk patients, 67/97 (69.1%) patients received Aza/Ven. Median survival time for all upfront induction regimens was 15.8 months (95% CI: 8.1-22.6), greatest median survival with Aza/Ven of 19.1 months (95% CI:10.4-23.6). Median PFS by all induction regimens was 8.9 months (95% CI: 5.0-13.9) with the greatest median PFS being Aza/Ven at 10.9 months (95% CI: 6.6-20.2); p=0.045). Overall, 29 out of 97 (29.9%) patients received salvage therapy. These included 19 (65.5%) males with 93.1% of patients being Caucasian. The majority of patients (81.5%) had adverse ELN risk stratification (Table 1). Total 1-year survival rate is 0.050 (0.31-0.67), with intensive 0.050 (0.16-0.67), low intensity 0.67 (0.35-0.86), targeted 0.39 (0.18-0.60). Total 3-year survival rate is 0.19 (0.06-0.38) with intensive 0.17 (0.02-0.46), low intensity 0.30 (0.08-0.56), and targeted 0.20 (0.05-0.41). Patients receiving subsequent salvage therapy after upfront venetoclax regimen showed the greatest PFS after receiving low-intensity therapy compared to targeted or intensive regimens with a median survival of 13.9 months (95% CI: 0.0-35.9); p<0.001) (Figure 1). Median survival after intensive therapy was 1.3 months (95% CI:0.5-3.9) and targeted therapy 5.8 months (95% CI: 2.8-10.0). Total OS 14.9 (95% CI: 6.1-22.7) with a low-intensity median survival of 22.7 months (95% CI:1.4-Not Reached (NR)). Conclusion: In this single-center retrospective review, it was found that a low-intensity salvage regimen after the upfront venetoclax regimen showed the greatest PFS compared to targeted or intensive regimens, however, there is no statistically significant difference in OS. Our study results were limited by a small sample size. Further studies are needed to clearly define the ideal salvage regimen for AML patients failing upfront venetoclax-based therapy.