Clinical Significance of Mitochondrial DNA Content in Acute Promyelocytic Leukemia

Used in the clinical practice for more than three decades, the all-trans retinoic acid (ATRA) rendered acute promyelocytic leukemia (APL) the most curable subtype of acute myeloid leukemia, and currently, its combination with arsenic trioxide (ATO) exceeded all expectations for a chemotherapy-free protocol. In terms of metabolic importance, ATRA can also modulate the mitochondria-mediated cellular metabolism and promote a shift from a glycolytic-driven metabolism to an oxidative phosphorylation profile, although this effect has never been demonstrated in APL. As part of the cellular metabolic machinery, mitochondrial DNA (mtDNA) content has been reported to be altered in different types of solid tumors with clinical implication on patient treatment outcomes, although its clinical significance in acute leukemias has not been investigated to the same extent. Particularly in acute promyelocytic leukemia (APL), the role of mtDNA content on prognostication is completely unknown. Considering that mostly APL samples display a glycolytic-driven metabolism, it is conceivable that APL patients harboring high mtDNA content may present a better response to ATRA-based therapies. To test this hypothesis, we determined the mtDNA content in samples from patients with APL enrolled in the International Consortium on Acute Promyelocytic Leukemia study (Rego et al. Blood. 2013 Mar 14;121(11):1935-43) and analyzed its relationship to treatment outcomes. Diagnostic bone marrow (BM) mononuclear cells from 156 consecutive patients with APL (median age: 35 years, range: 18-82 years; 45% male) were obtained at diagnosis. For comparison purposes, we also included peripheral blood (PB) from 293 age- and sex-adjusted healthy volunteers. First, we determined whether mtDNA content could be compared between PB mononuclear cells and BM. To do so, we measured the mtDNA content of 22 APL patients, for whom paired samples were available at the time of diagnosis and detected a strong correlation between PB and BM samples (Pearson correlation coefficient, r=0.78, 95% confidence interval, CI: 0.54 to 0.9). Next, we used the values of mtDNA higher than the 95 th percentile of healthy subjects (≥1.63. Note: this value represents a fold change relative to healthy control) to define APL patients with high mtDNA content. Patients that presented values within the range of normal control samples (<1.63) were classified as normal mtDNA content. The median follow-up among survivals was 40 months (95%CI: 34-47 months). Of the 131/156 patients who achieved complete remission, 18 patients (14%) relapsed. mtDNA content had no impact on complete remission achievement (84% for normal mtDNA versus 83% for high mtDNA; P=0.924) or overall survival (78% for normal mtDNA versus 80% for high mtDNA; P=0.69). In contrast, patients with high mtDNA content had a significantly high 5-year disease-free survival rate (86%, 95%CI: 78-95%) than patients with normal mtDNA content (61%, 95%CI: 46-82%). Considering non-relapse death as a competing cause of failure, the 5-year cumulative incidence of relapse (CIR) for patients with high and normal mtDNA content were 35% (95%CI: 16-49%) and 10% (95%CI: 2-17%), respectively. The multivariate Cox proportional hazards model showed that mtDNA content was independently associated with CIR (hazard ratio, HR: 0.31, 95%CI: 0.12-0.8) considering PETHEMA/GIMEMA risk of relapse subgroups and age as confounders. To functionally evaluate the metabolic alterations in APL cells upon ATRA treatment, NB4 cell line was treated with ATRA (1 µM) for 48 and 72 hours. In vitro analyses demonstrated (as expected) that the treatment with resulted in increased levels of myeloid maturation markers (CD11b/CD11c/CD15), with morphological changes being only observed at 72 hours. Metabolically, we observed an increase in mitochondrial mass and potential upon ATRA-treatment after 48 hours, which was also reflected by increase in the mtDNA content (2-fold increase in comparison with the vehicle). Together, these findings demonstrate an important, but not completely understood role for mtDNA content in APL. Disclosures Silveira: BMS/Celgene: Research Funding; Servier/Agios: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Pagnano: EMS: Other: Lecture; Jansenn: Other: Lecture; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture.

All-trans retinoic acid induces synaptopodin-dependent metaplasticity in mouse dentate granule cells

Previously we showed that the vitamin A metabolite all-trans retinoic acid (atRA) induces synaptic plasticity in acute brain slices prepared from the mouse and human neocortex (Lenz et al., 2021). Depending on the brain region studied, distinct effects of atRA on excitatory and inhibitory neurotransmission have been reported. Here, we used intraperitoneal injections of atRA (10 mg/kg) in adult C57BL/6J mice to study the effects of atRA on excitatory and inhibitory neurotransmission in the mouse fascia dentata—a brain region implicated in memory acquisition. No major changes in synaptic transmission were observed in the ventral hippocampus while a significant increase in both spontaneous excitatory postsynaptic current frequencies and synapse numbers were evident in the dorsal hippocampus 6 hr after atRA administration. The intrinsic properties of hippocampal dentate granule cells were not significantly different and hippocampal transcriptome analysis revealed no essential neuronal changes upon atRA treatment. In light of these findings, we tested for the metaplastic effects of atRA, that is, for its ability to modulate synaptic plasticity expression in the absence of major changes in baseline synaptic strength. Indeed, in vivo long-term potentiation (LTP) experiments demonstrated that systemic atRA treatment improves the ability of dentate granule cells to express LTP. The plasticity-promoting effects of atRA were not observed in synaptopodin-deficient mice, therefore, extending our previous results regarding the relevance of synaptopodin in atRA-mediated synaptic strengthening in the mouse prefrontal cortex. Taken together, our data show that atRA mediates synaptopodin-dependent metaplasticity in mouse dentate granule cells.

All-trans retinoic acid induces synaptopodin-dependent metaplasticity in mouse dentate granule cells

The vitamin A derivative all-trans retinoic acid (atRA) is a key mediator of synaptic plasticity. Depending on the brain region studied, distinct effects of atRA on excitatory and inhibitory neurotransmission have been reported. However, it remains unclear how atRA mediates brain region-specific effects on synaptic transmission and plasticity. Here, we used intraperitoneal injections of atRA (10 mg/kg) in adult male C57BL/6J mice to study the effects of atRA on excitatory and inhibitory neurotransmission in the mouse fascia dentata. In contrast to what has been reported in other brain regions, no major changes in synaptic transmission were observed in the ventral and dorsal hippocampus 6 hours after atRA administration. Likewise, no evidence for changes in the intrinsic properties of hippocampal dentate granule cells was obtained in the atRA-treated group. Moreover, hippocampal transcriptome analysis revealed no essential changes upon atRA treatment. In light of these findings, we tested for the metaplastic effects of atRA, i.e., for its ability to modulate synaptic plasticity expression in the absence of major changes in baseline synaptic transmission. Indeed, in vivo long-term potentiation (LTP) experiments demonstrated that systemic atRA treatment improves the ability of dentate granule cells to express LTP. The plasticity-promoting effects of atRA were not observed in synaptopodin-deficient mice, thus extending our previous results on the relevance of synaptopodin in atRA-mediated synaptic strengthening in the mouse prefrontal cortex. Taken together, our data show that atRA mediates synaptopodin-dependent metaplasticity in mouse dentate granule cells.

Regulatory Action of all trans Retinoic Acid on Metastasis Induced lung Cell Metabolic Changes during Implantation of B16F10 Cancer Cells in C57BL6 Mice

The changes that occur during metastasis lodging is under intense research now to develop preventive new drugs to fight against the deadly metastasis. The molecular drug, all trans Retinoic Acid (ATRA) has regulatory effects on signal mediated metabolism. In this study, we have analyzed the metastasis facilitating metabolic changes in mice lung when a highly metastatic melanoma cell line (B16F10) having potency to lodge in lung was implanted via tail vein injection into C57BL/6 mice (1×106 cells/ml in PBS). One group of implanted mice were treated with 0.60 mg of ATRA per Kg body weight daily for 21 days. The alteration of protein, enzymatic and non-enzymatic antioxidants (SOD, Catalase, GPX, GSH) levels and the lipid profile with cholesterol level were evaluated in the lung tissues. The ATRA treatment caused 62.16% inhibition on metastatic nodule formation. Compared to normal mice, the cancer control mice showed an increased (p≤ 0.01**) total protein, LPO and NO and a decreased antioxidant. In ATRA treated group, all these levels were reverted to near normal levels with a high significance (p≤ 0.01**) difference from untreated cancer mice. The lipid profile and cholesterol level also were altered in cancer and were normalized in ATRA treated group with high significance (p≤ 0.01**). All these results implies that the metabolic changes induced in the lung tissue during metastatic lodging of melanoma cells were prevented and regularized by the ATRA treatment in vivo which give a scope of anti-metastatic therapy using ATRA.

Thrombocytosis in a patient with acute promyelocytic leukemia during treatment with all-trans retinoic acid and arsenic trioxide

Thrombocytosis, an uncommon side effect of all-trans retinoic acid (ATRA) treatment, occurs in some patients with acute promyelocytic leukemia. Our case showed thrombocytosis on day 26 to day 32 of ATRA and arsenic trioxide therapies and then started to decrease gradually without changing ATRA dosage. Thrombocytosis may associate with cytokine.

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

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.

PML-RAR Binds to the +7kb Enhancer of CEBPE and Inhibits Its Expression

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.

ATRA Augments BCMA Expression on Myeloma Cells and Enhances Recognition By BCMA-CAR T-Cells

Background: B cell maturation antigen (BCMA) is a B-lineage antigen that is retained on malignant plasma cells in multiple myeloma (MM), and is under investigation as a target antigen for humoral and cellular immunotherapy. Targeting BCMA with chimeric antigen receptor (CAR) T-cells, T-cell engaging antibodies and antibody-drug conjugates has resulted in high rates of clinical responses however, the depth and durability of these responses is still not satisfactory and most patients ultimately relapse. This has been attributed at least in part to low or non-uniform BCMA expression on MM cells, as well as MM cell escape after BCMA down-regulation or even loss. Here, we show that epigenetic modulation with all-trans retinoic acid (ATRA) augments BCMA expression at the gene (and protein) level and leads to enhanced BCMA molecule density on the surface of MM cells that translates into increased anti-MM potency of BCMA CAR T-cells. Methods: Primary MM cells and myeloma cell lines were treated with titrated doses of ATRA (25, 50, 100 nM), alone and in combination with the g-secretase inhibitor crenigacestat (10 nM). BCMA expression was analyzed by flow cytometry, RT-qPCR and direct stochastic optical reconstruction microscopy (dSTORM). BCMA CAR T-cells were derived from healthy donors and MM patients (n&gt;6) and their anti-MM function analyzed in vitro and in the NSG/MM.1S murine xenograft model in vivo. Results: By RT-qPCR, we observed a 1.8-fold (MM.1S) and 2.1-fold (OPM-2) increase in BCMA gene expression after treatment with 50 nM ATRA for 72 hours. By flow-cytometry, we confirmed increased BCMA protein expression, with 1.9-fold (MM.1S and OPM-2) increase in mean fluorescence intensity relative to isotype control staining. Super-resolution dSTORM microscopy on MM.1S cells confirmed the increase in BCMA protein expression and showed a homogenous distribution pattern of BCMA molecules across the cell surface without an increase in cluster formation. These data were confirmed with primary MM cells from patients with newly diagnosed (n=7) and relapsed/refractory (n=11) MM. The increase in MFI for BCMA expression on primary MM cells after ATRA treatment was 1.2-fold - 2.2-fold (mean: 1.6-fold; p=.01 at 50 nM ATRA). By ELISA, we did not detect increased levels of soluble BCMA protein in supernatant of MM.1S cells after ATRA treatment. Accordingly, we found superior cytolytic activity, cytokine secretion and proliferation of CD8+ and CD4+BCMA CAR T-cells in response to ATRA-treated vs. non-treated primary MM cells and MM cell lines. In the NSG/MM.1S xenograft model, we confirmed increased BCMA expression on MM.1S after systemic treatment with ATRA, and superior anti-MM activity after adoptive transfer of BCMA CAR T-cells. Further, we confirmed that epigenetic modulation of BCMA-expression with ATRA works synergistically with g-secretase inhibitor treatment that has recently been shown to prevent cleavage of BCMA molecules from the surface of MM cells (Pont Blood 2019). Combination treatment with ATRA and the g-secretase inhibitor crenigacestat led to higher BCMA density on primary MM cells (and cell lines) than each single-agent treatment alone, resulting in maximum reactivity of by BCMA CAR T-cells in vitro and in vivo. Conclusions: Taken together, the data show that BCMA expression on MM cells can be increased by epigenetic modulation with ATRA. After ATRA treatment, MM cells have increased susceptibility to BCMA CAR T-cell treatment in pre-clinical models vitro and in vivo, that can be increased even further by combination treatment of ATRA and g-secretase inhibitors. These data suggest the potential to improve responses (depth and durability) of immunotherapies directed against BCMA. Disclosures Einsele: Takeda: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau.

RAR Gamma Activation Sensitizes Human Myeloma Cells to Carfilzomib Treatment through OAS-RNase L Innate Immune Pathway

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) kill MM cells by disrupting the degradation of misfolded proteins, presumably derived from high-level immunoglobulin production, which provides an appealing explanation for why MM cells are so uniquely sensitive to PIs. However, relapses are frequent and acquired resistance to PI treatment emerges in most patients. Therefore, identifying novel and safe drugs overcoming PI resistance in MM will aid in chemo-(re)sensitization, reducing PI-induced side effects, and maximizing the outcomes of PI therapy. Here we performed a high-throughput screen of 1855 FDA-approved drugs (Figure 1) and identified all-trans retinoic acid (ATRA), a classic pan-retinoic acid receptor (RAR) agonist used successfully to treat acute promyelocytic leukemia (APL), as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and re-sensitized Cfz-resistant MM cells to Cfz in vitro. To determine which RARs are important for ATRA enhancement of Cfz-induced apoptosis in MM, esiRNAs of RARs for knocking down RARs and selective agonists of RARs were used in Cfz-treated MM cells. We identified that RARγ activation is important for ATRA sensitizing MM cells to Cfz treatment. To determine which signaling pathways are involved in ATRA-treated MM cells, gene-profiling data analysis of Cfz- versus ATRA+Cfz-treated MM cells was performed and real-time PCR was used to validate the microarray results. We found that ATRA treatment activated IFN-β response pathway (Figure 2), leading to upregulated expression of IRF1 and OAS1-3. Interestingly, similar to ATRA, IFN-β, which alone did not induce MM apoptosis, enhanced Cfz-induced MM cell apoptosis. Furthermore, using RNA integrity assay and dsRNA detection assay, we identified that ATRA treatment elevated the expression of OASs, which synthesized 2-5A upon binding to dsRNA induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death (Figure 3). By knocking down each gene of OAS1-3, we demonstrated that OAS1 is essential for ATRA to sensitize MM cells to Cfz-induced apoptosis. Furthermore, we determined the impact and significance of RARγ and OAS1 in human MM pathogenesis and drug response by analyzing the gene-profiling data of 264 MM patients from Mulligan et al. datasets and 1,143 MM patients from MMRF coMMpass study IA13. In support of these findings, analyses of the large patient's gene-profiling datasets showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment (Figure 4). Finally, BMS961, a selective RARγ agonist, similar to ATRA, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo (Figure 5). Thus, this study highlights the potential for RARγ agonists to sensitize MM and overcome MM resistance to Cfz treatment in patients. Disclosures No relevant conflicts of interest to declare.

Interleukin-8 is not a predictive biomarker for the development of the acute promyelocytic leukemia differentiation syndrome

Background: Differentiation syndrome (DS) is the main life-threatening adverse event that occurs in acute promyelocytic leukemia (APL) patients treated with all-trans retinoic acid (ATRA). Cytokine imbalances have been reported to play role during the developing of acute promyelocytic leukemia differentiation syndrome (APL-DS). However, the relationship between the plasma cytokine levels and their prognostic value for the prediction of DS developing in patients with APL during the treatment with ATRA and anthracyclines has not been previously reported. Methods: In this study, we followed an APL cohort (n=17) over seven days of ATRA therapy in DS (n=6) and non-DS groups (n=11). Interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α were measured in the peripheral blood plasma from 17 patients with APL and 11 healthy adult controls by using the cytometric bead array method. Results: In non-DS patients, IL-8 plasma levels were significantly reduced in the seventh day of ATRA treatment (34.16; 6.99 to 147.11 pg mL-1 in D0 vs. 10.9; 0 to 26.81 pg mL-1 in D7; p = 0.02) whereas their levels did not discriminate between DS and non-DS development during the entire induction period (all p > 0.05 in D0, D3, and D7). No significant differences were found in IL-6 levels between groups (p > 0.05 in D0-D7). Other cytokines tested were all undetectable in patients with APL or healthy controls. Conclusions: We demonstrated that the modulation of IL-8 following ATRA treatment may occur regardless of the development of DS and, therefore, does not appear to be a predictive biomarker to monitor the APL-DS.