Immunotherapy with pVAX14 Nucleic Acid in Combination with All-Trans Retinoic Acid (ATRA) in an Acute Promyelocytic Leukemia (APL) Mouse Model Activates Cytotoxic T-Cells and Enhances Immune Responses: A Strategy for Turning “Cold” Tumors “Hot”

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.