Primary Acute Promyelocytic Leukemia Research Articles

Clinical characteristics and analysis of related factors associated with early death in newly diagnosed patients with acute promyelocytic leukemia

ObjectiveThe objective of this study was to investigate the clinical characteristics and analysis of related factors associated with early death in newly diagnosed patients with acute promyelocytic leukemia (APL).MethodsThis retrospective study included patients who visited our hospital between January 2010 and August 2022 and were diagnosed with APL for the first time. We analyzed their clinical and laboratory characteristics and analysis of related factors associated with early death.ResultsA total of 269 patients with a primary diagnosis of APL were collected. The male to female ratio was 6:5, and the median age was 42 years (range 7–80). Among patients with initial APL diagnosis, there were 34 early deaths, resulting in an early mortality rate of 13%. The median time from diagnosis to death was 8.5 days (range 3–24). Comparative analysis of the clinical characteristics between patients who died early and those who did not, using a logistic regression model, revealed that age, white blood cell count (WBC) at initial diagnosis, and prolongation time of prothrombin time (PT) were independent risk factors for early death in patients with primary APL (P < 0.05). Comparing the clinical characteristics during hospitalization between the early death group and the non-early death group, it was observed that the daily mean of WBC during hospitalization was significantly higher in patients who died early than in those who did not (P < 0.001). Conversely, the daily mean of platelet count (PLT) was significantly lower in patients who died early compared to those who did not (P < 0.001). Furthermore, statistically significant differences were found in the mean daily infusion of PLT (P < 0.05), fibrinogen (Fib) (P < 0.05), and fresh frozen plasma (FFP) (P < 0.05) during hospitalization between patients who died early and those who did not. Specifically, the mean daily infusion of PLT and FFP was significantly higher in the early-death group than in the non-early-death group. Cerebral hemorrhage was identified as the immediate cause of death in 25 out of the 34 early-death patients (74%). The remaining causes of death included infection in 5 cases (15%), all of which were severe pulmonary infections, including 2 cases of combined differentiation syndrome, and abandonment of treatment in 4 patients (11%) at initial diagnosis.ConclusionIn patients with primary APL, age, WBC at initial diagnosis, and PT prolongation time were identified as independent risk factors for early death (P < 0.05). Laboratory findings regarding WBC and PLT during hospitalization, as well as the infusion of PLT, Fib, and FFP during hospitalization, were also statistically significant. Cerebral hemorrhage was found to be the main cause of early death in patients with primary APL.

Successful treatment of acute promyelocytic leukemia with initial hyperleukocytosis, differentiated syndrome and massive intracranial hemorrhage in a child

Introduction. The development of hemorrhagic syndrome at the onset of the disease in patients with acute promyelocytic leukemia (APL) is one of the main causes of therapy failures and the most common cause of death in the early stages of remission induction. The risk of severe hemorrhagic complications increases in patients with the microgranular APL variant, often accompanied with hyperleukocytosis.Aim — to present a clinical case of successful diagnosis and treatment of primary APL with initial hyperleukocytosis in a patient with massive intracranial hemorrhage and differentiated syndrome.Main findings. A clinical observation of the diagnosis and treatment of APL in a 13-year-old patient with initial hyperleukocytosis, massive intracranial hemorrhage and differentiation syndrome is presented. Data on the variable manifestations of hemorrhagic syndrome in patients with APL and its effect on survival outcomes are also given. Diagnostic criteria and approaches in the treatment of differentiation syndrome are presented on the example of a clinical observation, when early clinical diagnosis and timely initiation of special antitumor treatment in the intensive care unit made it possible to achieve clinical, hematological and molecular remission, avoiding the fatal consequences of massive intracranial hemorrhage.

Phase separation of PML/RARα and BRD4 coassembled microspeckles governs transcriptional dysregulation in acute promyelocytic leukemia

In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARα) fusion protein destroys PML nuclear bodies (NBs), leading to the formation of microspeckles. However, our understanding, largely learned from morphological observations, lacks insight into the mechanisms behind PML/RARα-mediated microspeckle formation and its role in APL leukemogenesis. This study presents evidence uncovering liquid-liquid phase separation (LLPS) as a key mechanism in the formation of PML/RARα-mediated microspeckles. This process is facilitated by the intrinsically disordered region containing a large portion of PML and a smaller segment of RARα. We demonstrate the coassembly of bromodomain-containing protein 4 (BRD4) within PML/RARα-mediated condensates, differing from wild-type PML-formed NBs. In the absence of PML/RARα, PML NBs and BRD4 puncta exist as two independent phases, but the presence of PML/RARα disrupts PML NBs and redistributes PML and BRD4 into a distinct phase, forming PML/RARα-assembled microspeckles. Genome-wide profiling reveals a PML/RARα-induced BRD4 redistribution across the genome, with preferential binding to super-enhancers and broad-promoters (SEBPs). Mechanistically, BRD4 is recruited by PML/RARα into nuclear condensates, facilitating BRD4 chromatin binding to exert transcriptional activation essential for APL survival. Perturbing LLPS through chemical inhibition (1, 6-hexanediol) significantly reduces chromatin co-occupancy of PML/RARα and BRD4, attenuating their target gene activation. Finally, a series of experimental validations in primary APL patient samples confirm that PML/RARα forms microspeckles through condensates, recruits BRD4 to coassemble condensates, and co-occupies SEBP regions. Our findings elucidate the biophysical, pathological, and transcriptional dynamics of PML/RARα-assembled microspeckles, underscoring the importance of BRD4 in mediating transcriptional activation that enables PML/RARα to initiate APL.

Targeting HDAC3 to Overcome the Resistance to ATRA or Arsenic in Acute Promyelocytic Leukemia through Ubiquitination and Degradation of PML-RARα

Acute promyelocytic leukemia (APL) is driven by the oncoprotein PML-RARα, which recruits corepressor complexes, including histone deacetylases (HDACs), to suppress cell differentiation and promote APL initiation. All-trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) or chemotherapy highly improves the prognosis of APL patients. However, refractoriness to ATRA and ATO may occur, which leads to relapsed disease in a group of patients. In this study, we obtained the relationship between different HDACs with APL by analyzing transcriptional dataset of leukemia and confirmed the positive association of HDAC3 with PML-RARα in the mRNA and protein levels measured in primary human APL blasts. Experiments in vitro indicated that targeting HDAC3 but not HDAC1 or HDAC2 induced more effective differentiation and apoptosis in both NB4 cells and primary human APL cells. To examine the effects of HDAC3 inhibition on APL cells in vivo, we utilized normal FVB mice transplanted with APL cells from hMRP8-PML-RARα transgenic mice and NB4 or NB4-HDAC3Cas9 xenograft mouse model. We found that pharmacological or genetically inhibition of HDAC3 extended the survival rate of mice and alleviated spleen infiltration of leukemia cells and promoted the apoptosis and differentiation of APL cells. As to the molecular mechanism, we first examined whether HDAC3 affected PML-RARα expression level and the results demonstrated that HDAC3 inhibition reduced PML-RARα protein half-life rather than transcriptional activity via the ubiquitin proteasome system. In the subsequent experiments, we constructed a series of PML-RARα mutations. By confocal imaging system, acetylation and protein-protein interaction assays, we demonstrated that HDAC3 directly bound to the LBD and DBD domains of RARA protein and modulated the acetylation of lysine 394 of PML, which reduced PIAS1-mediated PML-RARα SUMOylation and subsequent RNF4-induced ubiquitylation. HDAC3 inhibition induced an increase of K394 acetylation, and promoted PML-RARα ubiquitylation and subsequent degradation, while the effect could be rescued by proteosome inhibition. Our results further revealed that the inhibition of HDAC3 induced degradation of both wild-type and ATRA- or ATO-resistant mutant PML-RARα (A216V, L218P, ΔF286 and R276Q). Therefore, we speculated that targeting HDAC3 could induce antileukemic effects on ATO- or ATRA-resistant APL cells. In NB4-R1, NB4-R2 and primary human APL cells, inhibition of HDAC3 increased significantly the cell differentiation and apoptosis, decreased PML-RARα expression and reduced the proliferation and colony-formation units (CFUs). For experiments in vivo, ATRA-resistant APL xenograft mouse model of NB4-R2 cells and patient-derived tumor xenograft (PDX) model were utilized to evaluate the effects of targeting HDAC3. Results showed that targeting HDAC3 extended the survival of mice, decreased the leukemia cells infiltration and increased the differentiation and apoptotic cells. In conclusion, our study identifies the role of HDAC3 as a positive regulator of the PML-RARα oncoprotein by deacetylating PML-RARα and suggests that targeting HDAC3 could be a promising strategy to treat relapsed/refractory APL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High MN1 Expression Is Associated with an Lspc-Enriched Phenotype and Glycolysis Representing a New Vulnerability in Acute Myeloid Leukemia

Despite a steady increase in the number of targeted therapies over the past decades, the 5-year overall survival (OS) rate remains poor in acute myeloid leukemia (AML). Currently, high expression of the oncogenic Meningioma 1 (MN1) is associated with poor OS in AMLs with normal karyotype, and we correlated its expression with a more immature cellular composition in AML samples. Hence, we hypothesized that supplementation of AML cells that usually fail to engraft in xenograft models, such as acute promyelocytic leukemia (APL), with MN1 would promote in vivo expansion in mice allowing the study of potential therapeutic vulnerabilities. The in vitro experiments were performed in human AML cell lines, primary AML cells (n=39), and isolated healthy CD34+ cells. MN1-overexpression (OE) was lentivirally performed. Bioenergetic profiles were evaluated by a Seahorse analyzer. Gene expression was measured by RNA-sequencing/qPCR. Protein levels were measured by immunofluorescence microscopy. Viability and surface protein expression was evaluated by flow cytometry. For in vivo studies, eight-weeks old female NSGS mice were transplanted with MN1-OE primary APL blast cells, and mice were followed for 12 weeks. Cells from the mouse organs were isolated, analyzed, and sorted for human APL blasts (CD45+CD117+CD33+) for ex vivo studies. We show that genetic MN1-OE in APL cells resulted in increased cell survival upon cytotoxic therapy, and decreased sensitivity to ATRA-induced terminal differentiation in short and long term cultures (3/8 days). In healthy CD34+ cells, we observed that MN1-OE was able to increase cell proliferation and stemness properties in CMP-sorted (CD34+CD38+CD123+CD45RA-) but not in GMP-sorted cells (CD34+CD38+CD123+CD45RA+), showing in human models the results from Heuser et al., 2011 in murine MN1-model. Co-expression of MN1+PML-RARα in healthy CD34+ cells resulted in superior expansion with retention of the CD34 marker, in comparison with the single hits. At the molecular level, APL cells with MN1high displayed transcriptional programs associated with "HYPOXIA"/"HSC-UP", whereas MN1low levels correlated with "OXPHOS-UP"/"GMP-UP". Despite no evident differences in Seahorse analysis, MN1-OE cells exhibited increased levels of the glycolytic enzyme PDK1. Functionally, MN1-OE cells were more sensitive to glycolytic inhibitors 2,2-dichloroacetophenone (a PDK1 inhibitor) and 2-deoxyglucose, revealing glycolysis as a metabolic vulnerability in MN1high AMLs, which was further validated in primary AML samples. These data are in line with our previous report indicating that more immature AML subtypes typically display a more glycolytic metabolic profile (Erdem et al, 2022). MN1 is an intrinsically disordered protein containing a long polyQ-stretch that is required for transformation (Riedel et al, 2021). We questioned whether the expression of chaperone proteins would be necessary for the appropriate functioning of MN1. We observed that expression of the DNAJB6 isoform 2 (DNAJB6b) correlated with MN1 expression across a large panel of genetic subtypes of AML, including more immature AML subtypes, such as the TP53-mutant AML but also in APL. shRNA-induced knockdown in AML cells of DNAJB6b resulted in cytosolic retention of MN1, associated with reduced cell survival. In vivo, the patient-derived xenograft model showed engraftment of APL samples upon MN1-OE, with the colonization of secondary organs and splenomegaly, which was not observed in the control. Flow cytometry analysis revealed that MN1-engrafted mice displayed increased levels of APL blasts in comparison with control, which had a large proportion of mature myeloid cells (CD33+CD11b+). Ex vivo screen of engrafted cells revealed increased resistance to ATRA-induced differentiation in MN1-OE sorted blasts. We provide further evidence to support the role of MN1 as an oncogenic hit allowing in vivo studies of difficult-to-engraft AML subtypes. Functionally, high MN1 expression resulted in decreased differentiation, retention of stem-like properties, and metabolic shift towards a more glycolytic state. We demonstrated that DNAJB6b controls the cellular localization of MN1, with its downregulation associated with the loss of MN1 supportive functions. Finally, our findings suggest that targeting glycolysis can be leveraged as a therapeutically actionable mechanism for driving cell death in MN1high AMLs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

S252: M2-POLARIZED MACROPHAGES CONTROL LEUKEMIC STEM CELL FATE BY PROMOTING METABOLIC REPROGRAMMING

Background: Being a crucial part of the tumor microenvironment (TME) in solid tumors, tumor-associated macrophages are often associated with poor prognosis (Bruni et al. Nat Rev Cancer 2020). Yet, in acute myeloid leukemia (AML) the role of macrophages remains unclear. Aims: Here, we evaluated the impact of M2 macrophages in AML using a patient derived xenograft (PDX) model. Methods: To do so, we first injected 1x105 peripheral blood (PB) derived M2 macrophages into NSGS mice and next transplanted notoriously difficult to engraft primary Acute Promyelocytic Leukemia (APL) cells (1 x 106 cells - n=7 different patient samples) via the retro-orbital vein. Results: As a result, mice with co-injected human M2-macrophages developed full-blown leukemia with increased spleen weight in comparison with the control. Perhaps even more strikingly, ex vivo culture of APL and other favorable AML subtypes such as inv (16) or NPM1 mutant leukemic cells on M2-macrophages for 48h was sufficient to “train” these cells to engraft and induce fatal leukemia. Maintenance of self-renewal was shown in a secondary transplant assay and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays (LSC frequency: Control: 1/6x106 cells vs M2 pre-culture: 1/7.2x104 cells). To better understand the biological changes induced on leukemic blast when exposed to M2 macrophages, we performed an RNA sequencing analysis comparing AML/APL samples at diagnosis to cells that were “trained” (48h) on M2-macrophages or on MS5 mesenchymal bone marrow stromal cells, as a control. Gene ontology and gene set enrichment analysis on the genes up regulated upon M2 co-culture were significantly enriched for oxidative phosphorylation (OxPhos) signatures. Evaluation of functional respiration using seahorse measurements, confirmed the increase of oxygen consumption rate (OCR, basal and maximum) in primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5. The increase of basal and maximum OCR suggested enhanced mitochondrial metabolism, which prompted us to determine whether macrophages, similar to MSC cells, can transfer mitochondria to primary AML cells (van der Vlist et al. Neuron 2022). Flow cytometry analysis revealed an efficient mitochondrial transfer from M2 macrophages to leukemic blasts, which was more efficient compared to mitochondrial exchange from MS5 cells. Treatment with the CPT1A inhibitor Etomoxir (50 µM), prevented the gain in functional respiration and enhanced proliferation of AML cells when those were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures. These results suggest increased fatty acid oxidation to drive the OXPHO-like state in AML. Summary/Conclusion: Overall, we revealed that M2 macrophages can support leukemic growth of favorable AML subtypes that are commonly difficult to engraft in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter the biology of AML cells transforming a non-engraftable cell into a cell with high leukemic potential. In vitro we show that AML blast cells cultured on M2 macrophage adapt a more OxPhos-like state linked to intrinsic changes associated with increased uptake of mitochondria. In summary, our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions.

PRMT5-mediated RNF4 methylation promotes therapeutic resistance of APL cells to As2O3 by stabilizing oncoprotein PML-RARα.

Acute promyelocytic leukemia (APL) is a hematological malignancy driven by the oncoprotein PML-RARα, which can be treated with arsenic trioxide (As2O3) or/and all-trans retinoic acid. The protein arginine methyltransferase 5 (PRMT5) is involved in tumorigenesis. However, little is known about the biological function and therapeutic potential of PRMT5 in APL. Here, we show that PRMT5 is highly expressed in APL patients. PRMT5 promotes APL by interacting with PML-RARα and suppressing its ubiquitination and degradation. Mechanistically, PRMT5 attenuates the interaction between PML-RARα and its ubiquitin E3 ligase RNF4 by methylating RNF4 at Arg164. Notably, As2O3 treatment triggers the dissociation of PRMT5 from PML nuclear bodies, attenuating RNF4 methylation and promoting RNF4-mediated PML-RARα ubiquitination and degradation. Moreover, knockdown of PRMT5 and pharmacological inhibition of PRMT5 with the specific inhibitor EPZ015666 significantly inhibit APL cells growth. The combination of EPZ015666 with As2O3 shows synergistic effects on As2O3-induced differentiation of bone marrow cells from APL mice, as well as on apoptosis and differentiation of primary APL cells from APL patients. These findings provide mechanistic insight into the function of PRMT5 in APL pathogenesis and demonstrate that inhibition of PRMT5, alone or in combination with As2O3, might be a promising therapeutic strategy against APL.

Blockade of deubiquitinase YOD1 degrades oncogenic PML/RARα and eradicates acute promyelocytic leukemia cells

In most acute promyelocytic leukemia (APL) cells, promyelocytic leukemia (PML) fuses to retinoic acid receptor α (RARα) due to chromosomal translocation, thus generating PML/RARα oncoprotein, which is a relatively stable oncoprotein for degradation in APL. Elucidating the mechanism regulating the stability of PML/RARα may help to degrade PML/RARα and eradicate APL cells. Here, we describe a deubiquitinase (DUB)-involved regulatory mechanism for the maintenance of PML/RARα stability and develop a novel pharmacological approach to degrading PML/RARα by inhibiting DUB. We utilized a DUB siRNA library to identify the ovarian tumor protease (OTU) family member deubiquitinase YOD1 as a critical DUB of PML/RARα. Suppression of YOD1 promoted the degradation of PML/RARα, thus inhibiting APL cells and prolonging the survival time of APL cell-bearing mice. Subsequent phenotypic screening of small molecules allowed us to identify ubiquitin isopeptidase inhibitor I (G5) as the first YOD1 pharmacological inhibitor. As expected, G5 notably degraded PML/RARα protein and eradicated APL, particularly drug-resistant APL cells. Importantly, G5 also showed a strong killing effect on primary patient-derived APL blasts. Overall, our study not only reveals the DUB-involved regulatory mechanism on PML/RARα stability and validates YOD1 as a potential therapeutic target for APL, but also identifies G5 as a YOD1 inhibitor and a promising candidate for APL, particularly drug-resistant APL treatment.

Podoplanin Expression in the Bleeding Complications of Acute Promyelocytic Leukemias

Background: Acute promyelocytic leukemia (APL), characterized by the t(15;17) translocation, represents a favorable-risk subgroup of AML patients when treated with ATRA-based regimens. The leading cause of early death and the major unmet medical need in this disease are the bleeding complications, which are mainly attributed to aberrant expression of Tissue Factor (TF) and Annexin A2 (ANXA2) on leukemic promyelocytes, leading to disseminated intravascular coagulation and to hyperfibrinolysis, respectively. APL patients are also at increased risk of venous and arterial thromboses. The mechanisms underlying APL-associated hemostatic complications are not fully elucidated.Aims and Methods: We analyzed the transcriptome of 30 APL specimens comprised in the Leucegene 430 AML cohort, as well as normal hematopoietic cell populations (n=63) aiming to better understand the hemostasis-related transcriptomic landscape of this subgroup.Results: We identified podoplanin (PDPN) as the most significantly and differentially overexpressed gene in APL (median 2.6 vs 0 RPKM for APL and AML, respectively, q-value = 7.3 x 10-29) and determined that PDPN gene expression correlates with protein surface expression as assessed by flow cytometry on primary APL cells (r=0.89). TF (6.6 vs 1.6, p=1.8x10-9) and ANXA2 (142.8 vs 69.1, p=1.9x10-5) are also more expressed in APL, but less specifically than PDPN. Podoplanin is a glycoprotein that physiologically binds to its receptor, CLEC2, on platelets to induce platelet aggregation causing the separation of blood and lymphatic vessels during embryogenesis (Uhrin et al Blood 2010). As anticipated, we found that PDPN is not expressed in all studied sorted cell subpopulations from normal blood or bone marrow specimens, including in promyelocytes (median of 0 RPKM for all), indicating that platelets are most likely not exposed to PDPN in the adult vasculature and that this protein is ectopically expressed on APL promyelocytes.Next, we used lentiviral gene transfer to engineer OCI-AML5 cells, which do not express PDPN, to ectopically express this protein (AML5PDPN). When mixed with platelet-rich plasma (PRP), we found that AML5PDPN cells have much greater platelet-binding and activating capacity than AML5CTRL cells. Using light transmission aggregometry, only AML5PDPN cells, not AML5CTRL cells, could induce platelet aggregation at tested concentrations. These results indicate that PDPN expression on leukemia cells is sufficient to induce platelet binding, activation and aggregation. Using patient-derived cells, we similarly found that PDPN-expressing primary APL cells (n=3 samples), but not PDPN-negative APL (n=2) or AML (n=5) cells, have the capacity to bind and activate platelets and to induce platelet aggregation. Our results suggest that PDPN could contribute to APL-related thromboses, as was recently reported in brain tumors (Riedl et al Blood 2017).We next tested the hypothesis that PDPN expression on human myeloid blasts leads to platelet consumption, thrombocytopenia and bleeding in vivo . NSG mice were transplanted with either AML5CTRL or AML5PDPN cells and were monitored until they showed clinical signs of leukemia. Platelet counts rapidly and significantly dropped in the AML5PDPN-cohort at day 25 and beyond when compared to control animals (median 343 vs 1005 x 109/L, p < 0.0001). Mice platelet counts were inversely correlated with circulating PDPN load (r=-0.88) and animals with PDPN+ leukemias had markedly prolonged bleeding time (median > 15:00 vs 7:45, p = 0.0003).Finally, we hypothesized that ATRA reduces bleeding complications in patients, at least partially, by decreasing PDPN expression. We found that PDPN surface protein on primary APL promyelocytes is markedly decreased within the first 24 hours of ATRA treatment in vitro, and levels reached less than 10% on day 4 when compared to untreated cells (DMSO). Arsenic trioxide or dexamethasone treatment led to a modest or no reduction in PDPN expression, respectively.Conclusion: PDPN expression is specific to APL and, by inducing platelet binding, activation and aggregation, it contributes to platelet consumption and to bleeding complications. Our findings may also expose a new promising angle for the treatment of the early hemostatic complications found in APL patients. DisclosuresNo relevant conflicts of interest to declare.

Use of Arsenic Trioxide (As2O3 ) in the Treatment of Acute Promyelocytic Leukemia (APL): I. As2O3 Exerts Dose-Dependent Dual Effects on APL Cells

AbstractRecent clinical studies in China showed that As2O3 is an effective and relatively safe drug in the treatment of acute promyelocytic leukemia (APL). We found previously that As2O3 can trigger apoptosis of APL cell line NB4 cells, which is associated with downregulation of bcl-2 gene expression and modulation of PML-RARα chimeric protein. To further understand the mechanisms of this alternative therapy for APL, we investigated in this report the effects of a wide range of concentrations of As2O3 on cultured primary APL cells, all-trans retinoic acid (ATRA)-susceptible (NB4 cells) and ATRA-resistant (MR2 subclone) APL cell lines. The results indicated that As2O3 had dose-dependent dual effects on APL cells: inducing preferentially apoptosis at relatively high concentrations (0.5 to 2 μmol/L) and inducing partial differentiation at low concentrations (0.1 to 0.5 μmol/L). The rapid modulation and degradation of PML-RARα proteins, which was induced by As2O3 at 0.1 to 2 μmol/L, could contribute to these two effects. Bone marrow and peripheral blood examination showed that myelocyte-like cells, probably as a result of partial in vivo differentiation, and degenerative cells increased after 2 to 3 weeks of continuous in vivo As2O3 treatment when leukemic promyelocytes decreased. In conclusion, combination of induction of apoptosis and partial differention could be the main cellular mechanisms of As2O3 in the treatment of APL, and PML-RARα could play an important role in determining the specific effects of As2O3 on APL cells.

Second Primary Malignancy after Acute Promyelocytic Leukemia: A Population-Based Study.

Simple SummaryAcute promyelocytic leukemia (APL) is a rare and aggressive subtype of acute myeloid leukemia (AML). Since the introduction of all-trans-retinoic acid (ATRA) in APL management, the survival rate has increased substantially. However, there is evidence that retinoids might enhance tumor growth and the risk of secondary malignancies. The relationship between secondary cancer risk and APL treatment that includes ATRA is incompletely characterized. In this study, we investigated the risk factors associated with second primary malignancies after treatment of APL. Age ≥ 40 years at diagnosis of APL was significantly associated with an increased risk of second malignancies. Our findings suggest a potential carcinogenic role for ATRA in the salivary gland, liver, and soft tissue malignancies. Moreover, secondary tumors were significantly more frequent among patients with primary APL than in individuals with non-APL malignancies. Our finding suggests opportunities for surveillance for patients who completed treatment for APL.Acute promyelocytic leukemia (APL), is now highly curable with treatment approaches that include all-trans retinoic acid (ATRA). The high incidence of APL in the Hispanics suggests an association with genetic variants in this population. Information on second primary malignancies (SPMs) in patients with APL is limited. The Surveillance, Epidemiology, and End Results (SEER) database was used to interrogate whether the rate of SPMs in patients with APL was associated with ethnicity and/or ATRA treatment. Between 2000 and 2016, 116 cases of SPM were diagnosed among 4019 patients with APL. The mean age at diagnosis of primary APL was 53.9 years (±15.7 years), and the mean age at diagnosis of SPMs was 59.0 years (±14.5 years). Comparisons with 3774 APL survivors who did not develop SPMs revealed that age ≥40 years at diagnosis of APL (p < 0.001) and non-Hispanic white ethnicity (p = 0.025) were associated with SPMs in APL survivors. Salivary gland, liver, and soft tissue malignancies were significantly more common in patients with primary APL than in individuals with non-APL malignancies. A risk analysis comparing patients who had APL with patients who had non-APL AML suggests that SPMs after APL is associated with ATRA treatment. Therefore, patient follow-up after APL should focus on early diagnosis of SPMs.

Reduced SLIT2 is Associated with Increased Cell Proliferation and Arsenic Trioxide Resistance in Acute Promyelocytic Leukemia.

Simple SummaryIn solid tumors, the altered expression of embryonic genes such as the SLIT-ROBO family has been associated with poor prognosis, while little is known about their role in acute myeloid leukemia (AML). Previous studies reported frequent hypermethylation of SLIT2 mediated by the methyltransferase enzyme EZH2 and more recently the PML protein, which are commonly found to be aberrantly expressed in AML. Here, we aim to assess retrospectively the clinical relevance of the SLIT2 gene in acute promyelocytic leukemia, a homogenous subtype of AML. We demonstrated that reduced SLIT2 expression was associated with high leukocyte counts and reduced overall survival in different APL cohorts. STLI2 treatment decreased APL growth, while SLIT2 knockdown accelerated cell cycle progression and proliferation. Finally, reduced expression of SLIT2 in murine APL blasts resulted in fatal leukemia associated with increased leukocyte counts in vivo. These findings demonstrate that SLIT2 can be considered as a prognostic marker in APL, and a potential candidate for clinical studies of a more heterogeneous disease, such as AML.The SLIT-ROBO axis plays an important role in normal stem-cell biology, with possible repercussions on cancer stem cell emergence. Although the Promyelocytic Leukemia (PML) protein can regulate SLIT2 expression in the central nervous system, little is known about SLIT2 in acute promyelocytic leukemia. Hence, we aimed to investigate the levels of SLIT2 in acute promyelocytic leukemia (APL) and assess its biological activity in vitro and in vivo. Our analysis indicated that blasts with SLIT2high transcript levels were associated with cell cycle arrest, while SLIT2low APL blasts displayed a more stem-cell like phenotype. In a retrospective analysis using a cohort of patients treated with all-trans retinoic acid (ATRA) and anthracyclines, high SLIT2 expression was correlated with reduced leukocyte count (p = 0.024), and independently associated with improved overall survival (hazard ratio: 0.94; 95% confidence interval: 0.92–0.97; p < 0.001). Functionally, SLIT2-knockdown in primary APL blasts and cell lines led to increased cell proliferation and resistance to arsenic trioxide induced apoptosis. Finally, in vivo transplant of Slit2-silenced primary APL blasts promoted increased leukocyte count (p = 0.001) and decreased overall survival (p = 0.002) compared with the control. In summary, our data highlight the tumor suppressive function of SLIT2 in APL and its deteriorating effects on disease progression when downregulated.

Pan-HDAC inhibitor panobinostat, as a single agent or in combination with PI3K inhibitor, induces apoptosis in APL cells: An emerging approach to overcome MSC-induced resistance.

The emergence of the drugs targeting epigenetic alterations has brought an optimistic outlook for cancer patients and probably put an end into the devastating effects of the disease. Given to the prominent involvement of histone deacetylase (HDAC) enzymes in the formation of neoplastic nature of acute promyelocytic leukemia (APL), this study was aimed to evaluate the suppressive effect of pan-HDAC inhibitor panobinostat on both NB4 and primary APL patients cells, either in the context of mono- or co-culture with mesenchymal stem cells (MSC). Panobinostat effectively reduced the survival of APL cells; however, as compared to NB4, the viability of primary cells was inhibited at higher concentrations. Our results also showed that although HDAC inhibition could merely block the survival signals transduced from MSC, the presence of PI3K inhibitor could robustly reinforce panobinostat cytotoxicity; suggesting that MSC-induced activation of PI3K may attenuate, at least partly, the efficacy of HDAC inhibition in APL cells. In addition, cellular and molecular investigations on NB4 revealed that not only panobinostat induced p21-mediated G1 arrest and ROS-mediated apoptosis, but also exerted a superior cytotoxicity when combined with c-Myc and autophagy inhibitors. Last but not least, we found that panobinostat combined with arsenic trioxide (ATO) prompted a synergistic effect and provided an improved therapeutic value in NB4; proposing that the abrogation of HDAC using panobinostat might be a befitting approach in APL, either as a single agent or in a combined-modal strategy.

Reduced SLIT2 Are Associated with Increased Cell Proliferation and Arsenic Trioxide Resistance in APL Cells

Background: Recently, the SLIT/ROBO axis became a therapeutic target for a variety of non-cerebral solid tumors because of its relevance in the regulation of angiogenesis, inflammatory cell chemotaxis, tumor cell migration and metastasis. In acute myeloid leukemia (AML), lower levels of SLIT2 expression were associated with poorer prognosis in studies that excluded patients with acute promyelocytic leukemia (APL) of the analysis (Golos et al., Arch Immunol 2019). Our group previously reported that higher SLIT2 expression was associated with improved overall survival, disease-free survival and decreased cumulative incidence of relapse. Subsequently, our in vitro functional studies indicated anti-proliferative effects exercised by exogenous SLIT2 peptide treatment in APL (Weinhauser et al., Blood 2018). Although, the expression of SLIT/ROBO was detected on leukemic cells, the fact that in healthy subjects the main source of SLIT2 derives from bone marrow stromal cells (BMSC) (Smith-Berdan et al., Cell Cycle 2012) was so far neglected in the context of hematological malignancies. Aims: Here, we expanded our data on the role of SLIT2 using lentivirally knocked down APL cells, and addressed the issue of the importance of BMSC-derived SLIT2 on APL development. Methods: NB4 and NB4R2 (RA-resistant) and primary APL cells (age, 25-47y; n=4) were transduced with shRNA-SLIT2 or shCT (control). After synchronization using double-thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For the apoptosis analysis, cells were treated with ATO (1 µM) alone or in combination with RA (1 µM) for 24, 48 and 72 hours. The granulocytic differentiation in response to RA treatment alone (1 µM) or in combination with ATO (1 µM) for 48 and 72 hours, was evaluated based on the CD11b and CD11c levels. To assess the effect of SLIT2 silencing in BMSCs, HS5 and HS27A cell lines were transduced with shSLIT2 or shCT. Non-transduced NB4, NB4R2 and primary APL cells were co-cultured with engineered BMSCs and proliferation, apoptosis and granulocytic differentiation was performed in response to ATO alone or in combination with RA for 48 and 72 hours. In vivo assessment was performed using four 8-12 week-old C57/BL6PepBoy (CD45.1) lethally irradiated, transplanted with 5 x 106 hCG-PMLRARa blasts (CD45.2) transduced with shSlit2-murine and shCT. Results: Silencing SLIT2 in primary APL samples resulted in a significant increase in cell growth at the sixth day of culture compared to shCT samples (P&lt;.001). Accordingly, enhanced staining of Ki67 was detected in NB4 and NB4R2-shSLIT2 transduced compared to shCT cells. Moreover, NB4-shSLIT2 cells exhibited lower ATO-induced apoptosis either when ATO was used alone or in combination with RA. Nevertheless, SLIT2 knockdown had no effect on NB4R2 cells suggesting that RARA moiety of PML/RARA may be important to the observed effect. Concomitantly, NB4 cell differentiation rate, was greatly increased in NB4-shSLIT2 in the presence of RA after 48 and 72h (P&lt;.05) and 48h when RA was combined with ATO (P&lt;.05). In vivo transplantation of primary murine shSLIT2 hCG-PMLRARa blasts, resulted in significant increase of white blood cell count during the course of APL emergence and increased extramedullary hematopoiesis in the spleen, yet with no difference in the BM at the time of sacrifice. Next, we evaluated the number of transcripts and the function of SLIT2 in AML derived MSC cells. SLIT2 transcript levels were significantly reduced in primary AML-derived BMSC compared to BMSCs obtained from healthy donors (P&lt;.05). In vitro assays co-culturing NB4 and NB4R2 cells on engineered HS5 or HS27A BMSC cells showed no difference in drug-induced apoptosis or differentiation (P&gt;.05). In contrast to shSLIT2 APL induced cell growth, co-culture of primary APL blast cells with transduced HS27A cells had no impact on proliferation, while the expression of the neutrophilic differentiation gene ELANE2 was significantly decreased in APL cells co-cultured with HS27A-shSLIT2.ConclusionSLIT2 is expressed in APL cells and exerts an anti-proliferative effect. On the other hand, SLIT2 produced by BMSC seemed to promote APL cellular stemness. Our results reinforce that SLIT2 function is cell type dependent and may be involved in the cross-talk between microenvironment and leukemic cells. Disclosures Figueiredo-Pontes: Novartis: Honoraria.

Gata2 Expression and Function in PML-RARA-driven Preleukemia and APL

Self-renewal is a key feature of the cells that initiate acute myeloid leukemia. To identify the mechanisms involved in PML-RARA (PR)-driven self-renewal, we made use of Ctsg-PR mice, which have PR knocked into the UTR of the first exon of Ctsg. Ctsg-PR drives the expression of PR in myeloid progenitor cells and gives these cells the ability to serially replate in methylcellulose-based colony assays. Most Ctsg-PR mice develop acute promyelocytic leukemia (APL) with an average latency of ~300 days. To identify target genes regulated by Ctsg-PR, we performed single cell RNA-seq (scRNA-seq) on whole bone marrow from young, preleukemic Ctsg-PR mice or age-matched littermates. We identified 959 differentially expressed genes (DEGs) within myeloid progenitors (546 upregulated by PR, and 413 downregulated). Gata2 was identified as a DEG in this analysis, and we confirmed this phenotype with bulk RNA-seq of purified promyelocytes from young, preleukemic WT vs. Ctsg-PR mice. All APLs derived from Ctsg-PR mice also expressed high levels of Gata2. To identify the immediate-early target genes of PR, we transduced human CD34+ cord blood cells with MSCV-IRES-GFP retroviruses containing PR, a mutant PR with a RARA DNA binding mutation (C88A, known to abolish PR replating), or an empty vector. ScRNA-seq analysis of these cells after 7 days of ex vivo culture identified 1815 DEGs (1301 upregulated and 514 downregulated by PR) in GFP+ cells expressing PR, compared to GFP+ cells transduced with PRC88A or empty vector. Among the DEGs, Gata2 was upregulated 5-fold in the PR GFP+ cells. Identical short-term retroviral overexpression studies with mouse marrow revealed that PR expression caused an expansion of hematopoietic progenitor cells that overexpressed Gata2. Finally, although normal human promyelocytes do not express GATA2, virtually all primary human APL samples do. Combined, these studies strongly suggest that GATA2 is a target gene of PR, and may therefore play a role in the development of APL. Based on our expression data, we hypothesized that Gata2 inactivation would reduce PR-driven self-renewal. To test this hypothesis, we bred Ctsg-PR mice to Rosa26-Cas9 mice, which ubiquitously express Cas9. Marrow from the resulting Ctsg-PR x Cas9 mice was electroporated with guide RNAs (gRNAs) targeting the zinc finger 1 (ZF1) domain or exon 2 of Gata2, or control loci (Actb intron 5 or Rosa26 intron 1) and serially replated in methylcellulose with SCF, IL-3, and IL-6. CRISPR-Cas9 efficiently induced a wide array of indel mutations at all gRNA target sites. Two days after electroporation, the frequency of Gata2 alleles with indels at the target site ranged from 64% to 93% (n=4); hundreds of different Gata2 indels were generated in each experiment. To our surprise, the Gata2 targeted Ctsg-PR cells replated with dramatically higher efficiency than control locus targeted cells (Figure 1). The enhanced replating efficiency was dependent upon PR, since Gata2 targeted Rosa26-Cas9 bone marrow did not serially replate. Further, cells with Gata2 indels were positively selected for over time. For example, in one experiment, the frequency of a 12 bp deletion in Gata2 (that caused an in-frame deletion in ZF1) rose from an initial variant allele frequency (VAF) of 3% to 70% after 8 weeks of replating. A larger Gata2 deletion was co-selected at a similar frequency, and resulted in the deletion of Gata2 exon 4, which encodes 90% of ZF1. Virtually all of the positively selected cells contained Gata2 indels, demonstrating the competitive advantage of Gata2 loss-of-function mutations in this setting. Control gRNAs did not lead to significant changes in plating efficiency, nor were any indels selected for. Additionally, Ctsg-PR x Cas9 cells with Gata2 indels shifted from a neutrophilic phenotype (Gr1+ CD11b+) to a monocytic one (CD11b+ Gr1-) at late passages. To further investigate the role of Gata2 in Ctsg-PR induced APL, we sequenced the genomes of 16 mouse APLs and found that 2 samples had spontaneous mutations in Gata2 (R330L and N363fs, with VAFs of 74% and 39% respectively). In summary, these data provide evidence that PR positively regulates Gata2, and that Gata2 in turn promotes neutrophilic differentiation of committed, "reprogrammed" myelomonocytic progenitors. Surprisingly, Gata2 appears to contribute to the lineage fate and proliferative capacity of PR-expressing hematopoietic progenitor cells. Disclosures No relevant conflicts of interest to declare.

The genetics and clinical characteristics of children morphologically diagnosed as acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by t(15;17)(q22;q21), resulting in a PML-RARA fusion that is the master driver of APL. A few cases that cannot be identified with PML-RARA by using conventional methods (karyotype analysis, FISH, and RT-PCR) involve abnormal promyelocytes that are fully in accordance with APL in morphology, cytochemistry, and immunophenotype. To explore the mechanisms involved in pathogenesis and recurrence of morphologically diagnosed APL, we performed comprehensive variant analysis by next-generation sequencing in 111 pediatric patients morphologically diagnosed as APL. Structural variant (SV) analysis in 120 DNA samples from both diagnosis and relapse stage identified 95 samples with RARA rearrangement (including 94 with PML-RARA and one with NPM-RARA) and two samples with KMT2A rearrangement. In the eligible 13 RNA samples without any RARA rearrangement at diagnosis, one case each with CPSF6-RARG, NPM1-CCDC28A, and TBC1D15-RAB21 and two cases with a TBL1XR1-RARB fusion were discovered. These uncovered fusion genes strongly suggested their contributions to leukemogenesis as driver alternations and APL phenotype may arise by abnormalities of other members of the nuclear receptor superfamily involved in retinoid signaling (RARB or RARG) or even by mechanisms distinct from the formation of aberrant retinoid receptors. Single-nucleotide variant (SNV) analysis in 77 children (80 samples) with RARA rearrangement showed recurrent alternations of primary APL in FLT3, WT1, USP9X, NRAS, and ARID1A, with a strong potential for involvement in pathogenesis, and WT1 as the only recurrently mutated gene in relapsed APL. WT1, NPM1, NRAS, FLT3, and NSD1 were identified as recurrently mutated in 17 primary samples without RARA rearrangement and WT1, NPM1, TP53, and RARA as recurrently mutated in 9 relapsed samples. The survival of APL with RARA rearrangement is much better than without RARA rearrangement. Thus, patients morphologically diagnosed as APL that cannot be identified as having a RARA rearrangement are more reasonably classified as a subclass of AML other than APL, and individualized treatment should be considered according to the genetic abnormalities.

Slit-Robo Pathway Is Clinically Relevant and May Represent a Potential Target in Acute Promyelocytic Leukemia

Slit-Robo Pathway Is Clinically Relevant and May Represent a Potential Target in Acute Promyelocytic Leukemia

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells.

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As2 O 3 (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As 2 O 3 (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.

Profiling and functional analysis of circular RNAs in acute promyelocytic leukemia and their dynamic regulation during all-trans retinoic acid treatment

Circular RNAs (circRNAs) are a novel class of powerful regulators in gene expression and participate in the pathogenesis of many diseases, including cancer. However, little is known about the roles of circRNAs in the development and treatment of acute promyelocytic leukemia (APL). Here we report the expression profiling and function of circRNAs in APL, including their dynamic regulation during all-trans retinoic acid (ATRA)-induced differentiation. We performed two independent ribosomal RNA-minus RNA-sequencing (Ribo-minus RNA-seq) experiments with and without RNase R treatment on APL patient-derived NB4 cells and identified a total of 4313 circRNAs, including 1098 newly identified circRNAs. Detailed analysis showed that circRNAs expressed in APL cells were mostly exon-derived, not by-products during splicing, and could be distinguished from hematopoietic stem cells, neutrophils and lymphocytes. The true presence and stability of circRNAs were verified both in NB4 cells and primary APL patient samples. Moreover, we conducted a time-series analysis of circRNAs on ATRA-treated NB4 cells and uncovered 508 circRNAs with dynamic expression during ATRA treatment, including 246 upregulated and 262 downregulated. Further evidence demonstrated that the majority of circRNAs were regulated independently of their host linear mRNAs. Detailed functional experiments demonstrated that circ-HIPK2, one of the differentially expressed circRNAs, significantly influenced ATRA-induced differentiation of APL cells. Further mechanistic studies revealed that circ-HIPK2 was located in cytoplasm and served as a sponge for differentiation-associated miR-124-3p. Finally, circ-HIPK2 expression in APL patients was significantly lower than that in normal peripheral mononuclear cells and other subtypes of AML, indicating its potential role as an APL biomarker. Our study indicates the biological functions of circRNAs in the development and treatment of APL, and provides a comprehensive circRNA resource for future studies.

Transcriptomic landscape of acute promyelocytic leukemia reveals aberrant surface expression of the platelet aggregation agonist Podoplanin.

Acute promyelocytic leukemia (APL) is a medical emergency because of associated lethal early bleeding, a condition preventable by prompt diagnosis and therapeutic intervention. The mechanisms underlying the hemostatic anomalies of APL are not completely elucidated. RNA-sequencing-based characterization of APL (n = 30) was performed and compared to that of other acute myeloid leukemia (n = 400) samples and normal promyelocytes. Perturbations in the transcriptome of coagulation and fibrinolysis-related genes in APL extend beyond known culprits and now include Thrombin, Factor X and Urokinase Receptor. Most intriguingly, the Podoplanin (PDPN) gene, involved in platelet aggregation, is aberrantly expressed in APL promyelocytes and is the most distinctive transcript for this disease. Using an antibody panel optimized for AML diagnosis by flow cytometry, we also found that PDPN was the most specific surface marker for APL, and that all-trans retinoic acid therapy rapidly decreases its expression. Functional studies showed that engineered overexpression of this gene in human leukemic cells causes aberrant platelet binding, activation and aggregation. PDPN-expressing primary APL cells, but not PDPN-negative primary leukemias, specifically induce platelet binding, activation and aggregation. Finally, PDPN expression on leukemia cells in a xenograft model was associated with thrombocytopenia and prolonged bleeding time in vivo. Together our results suggest that PDPN may contribute to the hemostatic perturbations found in APL.