Radiofrequency ablation remodels the tumor microenvironment and promotes systemic immunomodulation in pancreatic cancer

Background and Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by resistance to therapy. A major contributing factor to therapeutic failure is profound desmoplasia and a well-documented hypoxic tumor microenvironment (TME). In PDAC, several therapeutic approaches, including chemotherapy and radiation alone or combined with immune checkpoint inhibitors, have shown minimal therapeutic success, placing an imperative need for the discovery and application of innovative treatments. Endoscopic ultrasound guided radiofrequency ablation (EUS-RFA) is a promising immunomodulator therapy for PDAC. In this work, we hypothesized RFA promotes local and systemic stromal and immunomodulating effects that can be identified for new combination therapeutic strategies. Methods: To test our hypothesis, a syngeneic PDAC mouse model was performed by symmetrically injecting 100k murine KPC cells in bilateral flanks of C57BL/6 female mice. RFA treatment initiated when tumors reached 200-500 mm3 and was performed only in the right flank. The left flank tumor (non-RFA contralateral side) was used as a paired control for further analysis. Results: RFA promoted a significant reduction in tumor growth rate 4 days after treatment in RFA treated and non-RFA side contralateral tumors from treated mice when compared to controls. Histological analysis revealed a significant increase in expression of cleaved Caspase3 in RFA treated tumors. In addition, collagen deposition and CD31+ cells were significantly elevated in RFA side and non-RFA contralateral tumors from RFA treated mice. Proteome profiling showed changes in C5a and IL-23 in RFA responsive tumors, indicating a role of RFA in modulating intratumoral inflammatory responses. Conclusions: These data indicate RFA promotes local and systemic anti-tumor responses in a syngeneic mouse model of PDAC implicating RFA treatment for local tumors as well as metastatic disease. Keywords: tumor associated macrophages; IL-23; tumor vasculature; ablation induced necrosis

Model-Based Anticancer Effect of Botulinum Neurotoxin Type A1 on Syngeneic Melanoma Mice

In recent, Botulinum Neurotoxin A1 (BoNT/A1) has been suggested as a potential anticancer agent due to neuronal innervation in tumor cells. Although potential BoNT/A1’s mechanism of action for the tumor suppression has been gradually revealed so far, there were no reports to figure out the exposure-response relationships because of the difficulty of its quantitation in the biological matrix. The main objectives of this study were to measure the anticancer effect of BoNT/A1 using a syngeneic mouse model transplanted with melanoma cells (B16-F10) and developed a kinetic-pharmacodynamic (K-PD) model for quantitative exposure-response evaluation. To overcome the lack of exposure information, the K-PD model was implemented by the virtual pharmacokinetic compartment link to the pharmacodynamic compartment of Simeoni’s tumor growth inhibition model and evaluated using curve-fitting for the tumor growth-time profile after intratumoral injection of BoNT/A1. The final K-PD model was adequately explained for a pattern of tumor growth depending on represented exposure parameters and simulation studies were conducted to determine the optimal dose under various scenarios considering dose strength and frequency. The optimal dose range and regimen of ≥13.8 units kg−1 once a week or once every 3 days was predicted using the final model in B16-F10 syngeneic model and it was demonstrated with an extra in-vivo experiment. In conclusion, the K-PD model of BoNT/A1 was well developed to optimize the dosing regimen for evaluation of anticancer effect and this approach could be expandable to figure out quantitative interpretation of BoNT/A1’s efficacy in various xenograft and/or syngeneic models.

The multi-specific VH-based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity

Background Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. Methods CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. Results CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. Conclusions CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.

Spontaneous Physical Activity in Obese Condition Favours Antitumour Immunity Leading to Decreased Tumour Growth in a Syngeneic Mouse Model of Carcinogenesis

Our goal was to evaluate the effect of spontaneous physical activity on tumour immunity during aging. Elderly (n = 10/group, 33 weeks) ovariectomized C57BL/6J mice fed a hyperlipidic diet were housed in standard (SE) or enriched (EE) environments. After 4 weeks, orthotopic implantation of syngeneic mammary cancer EO771 cells was performed to explore the immune phenotyping in the immune organs and the tumours, as well as the cytokines in the tumour and the plasma. EE lowered circulating myostatin, IL-6 and slowed down tumour growth. Spleen and inguinal lymph node weights reduced in relation to SE. Within the tumours, EE induced a lower content of lymphoid cells with a decrease in Th2, Treg and MDCS; and, conversely, a greater quantity of Tc and TAMs. While no change in tumour NKs cells occurred, granzyme A and B expression increased as did that of perforin 1. Spontaneous physical activity in obese conditions slowed tumour growth by decreasing low-grade inflammation, modulating immune recruitment and efficacy within the tumour.

Indoleamine 2, 3-Dioxygenase Promotes Aryl Hydrocarbon Receptor-Dependent Differentiation Of Regulatory B Cells in Lung Cancer

Regulatory B cells (Breg) are IL-10 producing subsets of B cells that contribute to immunosuppression in the tumor microenvironment (TME). Breg are elevated in patients with lung cancer; however, the mechanisms underlying Breg development and their function in lung cancer have not been adequately elucidated. Herein, we report a novel role for Indoleamine 2, 3- dioxygenase (IDO), a metabolic enzyme that degrades tryptophan (Trp) and the Trp metabolite L-kynurenine (L-Kyn) in the regulation of Breg differentiation in the lung TME. Using a syngeneic mouse model of lung cancer, we report that Breg frequencies significantly increased during tumor progression in the lung TME and secondary lymphoid organs, while Breg were reduced in tumor-bearing IDO deficient mice (IDO-/-). Trp metabolite L-Kyn promoted Breg differentiation in-vitro in an aryl hydrocarbon receptor (AhR), toll-like receptor-4-myeloid differentiation primary response 88, (TLR4-MyD88) dependent manner. Importantly, using mouse models with conditional deletion of IDO in myeloid-lineage cells, we identified a significant role for immunosuppressive myeloid-derived suppressor cell (MDSC)-associated IDO in modulating in-vivo and ex-vivo differentiation of Breg. Our studies thus identify Trp metabolism as a therapeutic target to modulate regulatory B cell function during lung cancer progression.

The Krüppel-like Factor 4 Sustains a Leukemic Gene Expression Profile in MLL-AF9-Driven Acute Myeloid Leukemia

Among hematological malignancies, acute myeloid leukemia (AML) confers poor prognosis and limited progress has been made in the translation of decades of research into improved clinical outcomes. The current paradigm is that eradication of leukemia stem cells (LSCs) represents an avenue for overcoming relapse and refractory disease, but therapy focusing on eradicating this leukemic population has not been developed to-date. Further studies of unique signaling pathways and vulnerabilities in LSCs are warranted to design targeted therapies that could impact patient outcomes. To evaluate whether the stemness transcription factor Krüppel-like Factor 4 (KLF4) is important in the progression of AML, we retrovirally transduced MLL-AF9 into Klf4 fl/fl(fl/fl)and Klf4 fl/flVav-Cre (Δ/Δ) lineage − Sca-1 + c-Kit + (LSK) bone marrow cells and transplanted into C57BL/6 recipients. Here we report that the KLF4 promotes disease progression in the MLL-AF9-driven syngeneic AML mouse model. Strikingly, Δ/Δ AMLs exhibited improved disease latency and penetrance, and a seven-fold reduction in leukemia-initiating cell frequency in a secondary transplantation study. Δ/Δ LSCs, defined as leukemic granulocyte macrophage progenitors (L-GMP), demonstrated lessened clonogenicity in methylcellulose cultures and reduced representation of cells in the G 2/M phase of the cell cycle. RNAseq analysis of L-GMP revealed decreased expression of hematopoietic and leukemic stemness gene sets such as RAS signaling, and induction of inflammatory response gene (TNF-α, IFNα, IFNβ) pathways in Δ/Δ LSCs. To evaluate human relevance, we used CRISPR-Cas9 based targeted deletion of the human KLF4 gene in a MLL-AF9 PDX line and observed improved survival and defects in expansion as seen in the syngeneic mouse model . Lastly, to correlate KLF4-associated signaling present in murine AML LSCs with human AML, we used CRISPR-Cas9-based targeted deletion of KLF4 in MOLM-13 (KO) to generate two validated clones. MOLM-13 KO cells showed reduced cell proliferation in vitro and in vivo. Further, RPPA analysis revealed reduced RAS pathway activity (IR-β, β-Raf), accumulation of proteins associated with the S and G 1 phases (e.g., CDKN2A, p21, Histone H3, CENP-A), and decrease expression in regulators of the G 2/M checkpoint (e.g., Aurora A, B, Chk1, Plk1, Wee1, Cyclin B, pCDK1). Collectively, our data suggest a mechanism in which KLF4 contributes to AML disease by establishing a gene expression profile supporting stemness of AML LSCs. Disclosures No relevant conflicts of interest to declare.

Runx2 Deficiency in Osteoblasts Promotes Myeloma Resistance to Bortezomib By Increasing TSP-1-Dependent TGF-β1 Activation in Bone Marrow

Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib (BTZ) is one of the most effective front-line chemotherapeutic drugs for MM; however, 15-20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that MM cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OBs). In the current study, we investigated the impact of OB-Runx2 deficiency on the outcome of BTZ treatment using our syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 -/- mice) and OB-Runx2 +/+ mice as control. Five-week-old OB-Runx2 +/+ mice and OB-Runx2 -/- mice were i.v. injected with 5TGM1-Luc murine MM cells (2×10 6). On day 8, after the tumor injection, mice were randomly assigned to treatment for 4 weeks, with either BTZ (i.p. injection, 0.5 mg/kg body weight, twice/week) or PBS. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA showed that BTZ significantly inhibited tumor growth in OB-Runx2 +/+ mice, but not in OB-Runx2 -/- mice. Cytokine array and ELISA showed that in PBS-treated, tumor-bearing OB-Runx2 -/- mice, BM levels of thrombospondin-1 (TSP-1), a matricellular protein that converts latent TGF-β1 to its active form, and active TGF-β1 were significantly higher than levels in tumor-bearing OB-Runx2 +/+ counterparts. Interestingly, BTZ treatment further increased the levels of both TSP-1 and active TGF-β1 in the BM of tumor-bearing OB-Runx2 -/- mice, but it did not affect the level of either TSP-1 or TGF-β1 in the BM of tumor-bearing OB-Runx2 +/+ mice. These results suggest that OB-Runx2 deficiency increases TGF-β1 activation via TSP-1 in BM and BTZ treatment further enhances this effect. SRI31277 is a tripeptide antagonist that blocks TSP-1-mediated activation of TGF-β1 and it has been shown to reduce tumor burden, TGF-β signaling, and osteolytic bone disease in multiple mouse models of MM. To confirm the involvement of TSP-1/TGF-β1 activation in MM BTZ resistance induced by OB-Runx2 deficiency and to test whether blocking TSP1-mediated TGF-β1 activation can alleviate BTZ resistance, 5TGM1-Luc MM tumor-bearing OB-Runx2 +/+ and OB-Runx2 -/- mice were treated with PBS, BTZ, SRI31277 (osmotic pump, 30 mg/kg body weight per day), or BTZ + SRI31277 for 4 weeks. While SRI31277 did not further increase BTZ-induced MM inhibition in OB-Runx2 +/+ mice, SRI31277 treatment significantly reduced the tumor burden in OB-Runx2 -/- mice. Furthermore, treatment with BTZ + SRI31277 augmented the reduction in tumor burden induced by SRI31277 treatment alone. Flow cytometry and Western blot analyses demonstrated that SRI31277 treatment enhanced apoptosis, reduced Ki-67 expression as well as the activity of both canonical (SMAD2/3) and non-canonical (ERK1/2) signaling pathways of TGF-β1 in MM cells in OB-Runx2 -/- mice; and BTZ+SRI31277 treatment augmented these effects. Furthermore, TGF-β has immunosuppressive effects in MM and our data show that SRI31277 overcomes this by reducing myeloid derived suppressor cells, checkpoint, and T cell exhaustion markers and by increasing cytotoxic T cells. In conclusion, OB-Runx2 deficiency, induced by MM cells, promotes MM resistance to BTZ through the upregulation of TGF-β1 activation in the BM and TGF-β1 signaling in MM cells. Importantly, blocking TSP-1-mediated TGF-β1 activation with SRI31277 can reverse this resistance and immune dysfunction. Our findings demonstrate a novel mechanism for BTZ resistance in MM and identify a new target and strategy for overcoming immune dysregulation and chemoresistance in MM. Disclosures Murphy-Ullrich: Millipore Sigma: Patents & Royalties.