Background: While acute promyelocytic leukemia (APL) can generally be cured by current treatment (combining all-trans retinoic acid (ATRA) and arsenic (ATO)), it can serve as a model to develop immunotherapeutic approaches for other hematological malignancies. We have previously used an APL mouse model bearing the PML-RARA fusion gene to show that a non-specific immunotherapy comprising of a plasmid, pVAX14, coding for immunogenic open reading frames, which effectively code for neoantigens, in combination with ATRA, has a similar efficacy as a specific vaccine with PML-RARA fusion sequences with decreased bone marrow (BM) blasts, reduced minimal residual disease (PML-RARA transcripts) and measured immune responses to include an increase in anti-RARA antibody levels (Le Pogam et al Oncotarget 2015) - with long term survivors surviving up to 2 years (the mouse lifespan). The protective effect was shown to be T-cell mediated. In order to visualize cytotoxic killing we have imaged the effectors from immunized APL mice co-cultured with their APL targets in real time. Gene expression profiles revealed the activation of a gene list regulated in immune pathways. Methods: APL mice were treated with pVAX14 (3x100 micrograms every 20 days intramuscularly) + ATRA (5mg 21-day release). Effectors (total WBC or CD3+ T cells) were isolated from spleens of immunized mice and labelled (red) and co-cultured with APL BM cells labelled (green) (effector:target, E:T was 2:1) and visualized via timelapse imaging. Normal FVB/N BM were assayed as controls. Additional analyses were carried out by the incucyte Live-Cell Analysis Systems, which is like a flow cytometer that measures fluorescence in real time. Furthermore, APL mice were treated with ATRA + ATO (5 micrograms/g/mouse intraperitoneally for 28 consecutive days) without or with pVAX14 as previously described (Patel et al BCJ 2015). Bone marrows were harvested after the end of treatment and RNA-sequencing was performed on mice treated with ATRA+ATO (n=5) or ATRA + ATO + pVAX14 (n=7). Functional analysis using David identified immune related pathways. The genes regulated were ranked according to the frequency upregulated in the pathways, with confirmation of some by RQ-PCR. Results: Effectors originating from immunized mice were shown to kill APL BM target, sparing wild type BM cells. Two types of killing were observed, lytic or programmed cell death. Incucyte data showed an increase in apoptosis when the effectors were incubated with their APL targets compared with FVB/N BM marrow cells. Gene expression profiles showed distinct treatment signatures. Principle component analysis showed that the DNA treated mice had a more homogeneous expression pattern than mice without DNA treatment. The functional David pathway analysis identified 29 genes regulated in 16 immune pathways. RQ-PCR confirmed NLRP3 active in the inflammasome pathway as the most frequently regulated (13 pathways with >6-fold increased expression in the DNA treated mice relative to no DNA samples). Increased expression of Caspase 1 protein, also in the inflammasome pathway was additionally detected (3-fold increased expression in extracts from AML effectors co-cultured with AML targets compared with AML effectors with FVB/N targets). RQ-PCR also identified significant activation of innate immune pathway genes in immunized diseased mice compared to placebo such as the nucleic acid sensor, Hmgb1 (p=0.0002), the adapter molecule Myd88 (p=0.0017) and ATRA inducible Rig-I (p<0.0001). Conclusions: Visualization of cytotoxic T-cell kill in real time confirms one of the mechanisms of the protective effect of the immunotherapy. The gene list provides potential biomarkers of response to DNA treatment. Activation of the innate immune pathway suggests that this strategy can turn “cold” tumors “hot” and responsive to treatment. The findings using this APL model may be useful to treat other malignancies where the results of achieving remissions and cures are more challenging.

Differentially expressed genes in Xenograted T-ALL shSYNCRIP-SNHG5 versus ShCtrl

Supplementary Table S1 from Histone Deacetylase Inhibitors (HDI) Cause DNA Damage in Leukemia Cells: A Mechanism for Leukemia-Specific HDI-Dependent Apoptosis?

Supplementary Table 3 from Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Supplementary Figure 1 Legend from Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Supplementary Table 5 from Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Supplementary Table 4 from Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Differentially Expressed Proteins Deleted versus non-deleted clones

Supplementary Table 1 from Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Background: Due to their basal stress phenotype associated to transformation, cancer cells are addicted to non-mutated, non-oncogenic proteins that do not bear such vital functions in normal cells, a phenomenon referred as Non-Oncogenic Addiction (NOA). Targeting these NOAs could therefore induce selective killing of cancer cells, opening several therapeutic opportunities. Recent data suggest that the stress-related scaffold protein AAC-11 (anti-apoptosis clone 11, also known as Api5) is critically involved in cancer cells resistance to chemotherapies, metastatic potential and escape from the immune system. Methods: We have developed inactivating peptides based on the fusion of a cell penetrating sequence and portions of the leucine-zipper domain of AAC-11, which functions as a protein-protein interaction module. These peptides induce cancer cells death, through the inhibition of protein-protein interactions between AAC-11 and its partners, while sparing normal cells. We now describe further characterization of our lead peptide, JRT39, which contains residues 377 to 379 of AAC-11 linked to the cell-penetrating peptide “penetratin”. Results: In vitro, JRT39 causes cell death in a wide spectrum of cancer cell lines with IC50 ranging from 5 µM to 30 µM depending on tumor cell type. In particular, JRT39 showed selective efficacy towards primary cells from Acute Myeloid Leukemia (AML), Acute Lymphocytic Leukemia (ALL) or Sézary syndrome (SS) patients, while sparing normal hematopoietic cells, with an IC50 of 5-15 µM. Mechanistically, JRT39 induces membranolysis of cancer cells through binding to p21-activated kinase 1 (PAK1) in AML or SS cells plasma membrane, where PAK1 is overexpressed. In addition, JRT39 exerted potent anti-tumor activity in vivo in disseminated or subcutaneous AML, APL (Acute Promyelocytic Leukemia) and SS-patient derived preclinical murine models. Preliminary pharmacokinetic studies revealed that JRT39 is stable in human serum and has a plasma half-life of ~1.5-2.5 hours after intravenous (IV) administration to dogs or cynomolgus monkeys, with concentration-time data fitting 2-compartment model. Finally, JRT39 was well tolerated at 5 and 10 mg/kg after single or repeated (daily) IV injections. Conclusions: Combined, our preclinical data confirm that interfering with AAC-11-related survival pathways is a promising novel anticancer strategy and support the development of JRT39 for the treatment of cancer. Citation Format: Louise Jeammet, Emile Adicéam, Justine Habault, Anna Kaci, Jeannig Berrou, Mélanie Dupont, Nicolas Thonnart, Ewa Pasquereau-Kotula, Anne Marie-Cardine, Armand Bensussan, Marika Pla, Hervé Dombret, Claude Gardin, Martine Bagot, Jean-Christophe Rain, Hélène Sicard, Jérôme Tiollier, Thorsten Braun, Jean-Luc Poyet. AAC-11 survival pathways as therapeutic target in cancer: AAC-11 leucine-zipper domain derived peptides exert potent antitumor effects and exhibit favorable stability, pharmacokinetic and toxicology profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 464.

Supplementary Table 2 from Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Supplementary Figure 1 from Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Supplementary Procedures, Figures 1-4 from BCL-2 and Mutant NRAS Interact Physically and Functionally in a Mouse Model of Progressive Myelodysplasia

Supp Fig. S1-6, Supp Tables S1-S3, Supp Methods and References

Supplementary Table 4 from Reactive Oxygen Species, DNA Damage, and Error-Prone Repair: A Model for Genomic Instability with Progression in Myeloid Leukemia?

Differentially translated transcripts - translatome

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation. PML NBs recruit many partner proteins, but the actual biochemical mechanism underlying their pleiotropic functions remains elusive. Similarly, PML role in embryonic stem cell (ESC) and retro-element biology is unsettled. Here we demonstrate that PML is essential for oxidative stress-driven partner SUMO2/3 conjugation in mouse ESCs (mESCs) or leukemia, a process often followed by their poly-ubiquitination and degradation. Functionally, PML is required for stress responses in mESCs. Differential proteomics unravel the KAP1 complex as a PML NB-dependent SUMO2-target in arsenic-treated APL mice or mESCs. PML-driven KAP1 sumoylation enables activation of this key epigenetic repressor implicated in retro-element silencing. Accordingly, Pml-/- mESCs re-express transposable elements and display 2-Cell-Like features, the latter enforced by PML-controlled SUMO2-conjugation of DPPA2. Thus, PML orchestrates mESC state by coordinating SUMO2-conjugation of different transcriptional regulators, raising new hypotheses about PML roles in cancer.

SummaryMembrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation, but their role in pluripotency remains elusive. Here we establish that PML is required for basal SUMO2/3 conjugation in mESCs and oxidative stress-driven sumoylation in mESCs or in vivo. PML NBs create an oxidation-protective environment for UBC9-driven SUMO2/3 conjugation of PML partners, often followed by their poly-ubiquitination and degradation. Differential in vivo proteomics identified several members of the KAP1 complex as PML NB-dependent SUMO2-targets. The latter drives functional activation of this key epigenetic repressor. Accordingly, Pml−/− mESCs re-express transposable elements and display features of totipotent-like cells, a process further enforced by PML-controlled SUMO2-conjugation of DPPA2. Finally, PML is required for adaptive stress responses in mESCs. Collectively, PML orchestrates mESC fate through SUMO2-conjugation of key transcriptional or epigenetic regulators, raising new mechanistic hypotheses about PML roles in normal or cancer stem cells.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.