Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1). To address the critical need for novel therapeutics in MPNST, we aimed to establish an ex vivo 3D platform that accurately captured the genomic diversity of MPNST and could be utilized in a medium-throughput manner for drug screening studies to be validated in vivo using patient-derived xenografts (PDX). METHODS: Genomic analysis was performed on all PDX-tumor pairs. Selected PDX were harvested for assembly into 3D microtissues. Based on prior work in our labs, we evaluated drugs (trabectedin, olaparib, and mirdametinib) ex vivo and in vivo. For 3D microtissue studies, cell viability was the endpoint as assessed by Zeiss Axio Observer. For PDX drug studies, tumor volume was measured twice weekly. Bulk RNA sequencing was performed to identify pathways enriched in cells. RESULTS: We developed 13 NF1-associated MPNST-PDX and identified mutations or structural abnormalities in NF1 (100%), SUZ12 (85%), EED (15%), TP53 (15%), CDKN2A (85%), and chromosome 8 gain (77%). We successfully assembled PDX into 3D microtissues, categorized as robust (\u3e90% viability at 48 h), good (\u3e50%), or unusable (\u3c50%). We evaluated drug response to robust or good microtissues, namely MN-2, JH-2-002, JH-2-079-c, and WU-225. Drug response ex vivo predicted drug response in vivo, and enhanced drug effects were observed in select models. CONCLUSIONS: These data support the successful establishment of a novel 3D platform for drug discovery and MPNST biology exploration in a system representative of the human condition

MEK inhibition synergizes with TYK2 inhibitors in NF1-associated malignant peripheral nerve sheath tumors

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with limited treatment options and poor survival rates. About half of MPNST cases are associated with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Overexpression of TYK2 occurs in the majority of MPNST, implicating TYK2 as a therapeutic target. EXPERIMENTAL DESIGN: The effects of pharmacologic TYK2 inhibition on MPNST cell proliferation and survival were examined using IncuCyte live cell assays in vitro, and downstream actions were analyzed using RNA-sequencing (RNA-seq), qPCR arrays, and validation of protein changes with the WES automated Western system. Inhibition of TYK2 alone and in combination with MEK inhibition was evaluated in vivo using both murine and human MPNST cell lines, as well as MPNST PDX. RESULTS: Pharmacologic inhibition of TYK2 dose-dependently decreased proliferation and induced apoptosis over time. RNA-seq pathway analysis on TYK2 inhibitor-treated MPNST demonstrated decreased expression of cell cycle, mitotic, and glycolysis pathways. TYK2 inhibition resulted in upregulation of the MEK/ERK pathway gene expression, by both RNA-seq and qPCR array, as well as increased pERK1/2 levels by the WES Western system. The compensatory response was tested with dual treatment with TYK2 and MEK inhibitors, which synergistically decreased proliferation and increased apoptosis in vitro. Finally, combination therapy was shown to inhibit growth of MPNST in multiple in vivo models. CONCLUSIONS: These data provide the preclinical rationale for the development of a phase I clinical trial of deucravacitinib and mirdametinib in NF1-assosciated MPNST

Role of Calmodulin-Calmodulin Kinase II, cAMP/Protein Kinase A and ERK 1/2 on <i>Aeromonas hydrophila</i>-Induced Apoptosis of Head Kidney Macrophages

<div><p>The role of calcium (Ca²⁺) and its dependent protease calpain in <i>Aeromonas hydrophila</i>-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKII<i>g</i>) in the process. We observed significant increase in CaM levels in <i>A. hydrophila</i>-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca²⁺-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKII<i>g</i>. Using the CaMKII<i>g</i>-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKII<i>g</i> signalling to be critical for apoptosis of <i>A. hydrophila</i>-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKII<i>g</i> expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in <i>A. hydrophila</i>-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKII<i>g</i> in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of <i>A. hydrophila</i>. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca²⁺ levels initiated by <i>A. hydrophila</i> activates CaM and calpain-2; both pathways converge on CaMKII<i>g</i> which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.</p></div

PKA is pro-apoptotic in <i>A. hydrophila</i>-infected HKM.

<p>(A) HKM were transfected with CaMKII<i>g</i>-siRNA, PKACA-siRNA or scrambled siRNA then infected with <i>A. hydrophila</i> and PKACA mRNA expression detected by real time PCR 2 h p.i. HKM were pre-treated with H-89 or transfected with PKACA-siRNA or scrambled siRNA then infected with <i>A. hydrophila</i> and checked for (B) Hoechst 33342 positive cells and caspase-3 activity and (C) AV-PI staining 24 h p.i. (D) <i>A. hydrophila</i> viability was checked following either pre-incubation of HKM with H-89, co-incubation with 8-Br-cAMP or transfection with PKACA-siRNA. The intracellular bacterial number was determined by dilution plating on nutrient agar plate. Vertical bars represent mean ± SE (n = 6). <b>*</b>P<0.05, compared to HKM; <b>^γ</b>P<0.05, compared to HKM+Sc; <b>^#</b>P<0.05, compared to HKM+B; <b>^±</b>P<0.05, compared to HKM+Sc+B. HKM, control head kidney macrophage; HKM+Sc, HKM transfected with scrambled siRNA; HKM+B, HKM infected with <i>A. hydrophila</i>; HKM+Sc+B, HKM transfected with scrambled siRNA infected with <i>A. hydrophila</i>; HKM+CaMKII<i>g</i>-siRNA+B, HKM transfected with CaMKII<i>g</i>-siRNA infected with <i>A. hydrophila</i>; HKM+PKACA-siRNA+B, HKM transfected with PKACA-siRNA infected with <i>A. hydrophila</i>; HKM+H-89+B, HKM pre-treated with H-89 infected with <i>A. hydrophila</i>; HKM+8-Br-cAMP+B, HKM exposed to 8-Br-cAMP infected with <i>A. hydrophila</i>.</p

<i>A. hydrophila</i>-induced ERK 1/2 phosphorylation is PKA mediated and is pro-apoptotic in HKM.

<p>(A) HKM were transfected with PKACA-siRNA, scrambled siRNA or pre-treated with H-89, U 0126 then infected with <i>A. hydrophila</i> and checked for total ERK and pERK activity 24 h p.i. using EIA based kits. HKM were pre-treated with or without U 0126 then infected with <i>A. hydrophila</i> and checked for (B) Hoechst 33342 positive cells and caspase-3 activity and (C) AV-PI staining 24 h p.i. Vertical bars represent mean ± SE (n = 6). <b>*</b>P<0.05, compared to HKM; <b>^γ</b>P<0.05, compared to HKM+Sc; <b>^#</b>P<0.05, compared to HKM+B; <b>^±</b>P<0.05, compared to HKM+Sc+B. HKM, control head kidney macrophage; HKM+Sc, HKM transfected with scrambled siRNA; HKM+B, HKM infected with <i>A. hydrophila</i>; HKM+Sc+B, HKM transfected with scrambled siRNA infected with <i>A. hydrophila</i>; HKM+PKACA-siRNA+B, HKM transfected with PKACA-siRNA infected with <i>A. hydrophila</i>; HKM+H-89+B, HKM pre-treated with H-89 infected with <i>A. hydrophila</i>; HKM+U 0126+B, HKM pre-treated with U 0126 infected with <i>A. hydrophila</i>.</p

Gene sequences.

<p>Gene sequences.</p

CaMKII<i>g</i> is CaM-dependent and pro-apoptotic.

<p>HKM were pre-treated separately with KN-93, KN-92, STO-609 or transfected with CaMKII<i>g</i>-siRNA or scrambled siRNA then infected with <i>A. hydrophila</i> and checked for (A) Hoechst 33342 positive cells and caspase-3 activity and (B) AV-PI staining 24 h p.i. (C) HKM were transfected separately with CaM-siRNA, CaMKII<i>g</i>-siRNA or scrambled siRNA then infected with <i>A. hydrophila</i> and CaMKII<i>g</i> mRNA expression detected 2 h p.i. by real time PCR. (D) HKM were transfected with CaM-siRNA, CaMKII<i>g</i>-siRNA, scrambled siRNA or pre-treated with CMZ, KN-93, KN-92, calpain-2<i>i</i> for different time periods then infected with <i>A. hydrophila</i> and CaMKII<i>g</i> protein content detected 24 h p.i. using EIA kit. Vertical bars represent mean ± SE (n = 6). <b>*</b>P<0.05, compared to HKM; <b>^γ</b>P<0.05, compared to HKM+Sc; <b>^#</b>P<0.05, compared to HKM+B; <b>^±</b>P<0.05, compared to HKM+Sc+B. HKM, control head kidney macrophage; HKM+Sc, HKM transfected with scrambled siRNA; HKM+B, HKM infected with <i>A. hydrophila</i>; HKM+Sc+B, HKM transfected with scrambled siRNA infected with <i>A. hydrophila</i>; HKM+CaM-siRNA+B, HKM transfected with CaM-siRNA infected with <i>A. hydrophila</i>; HKM+CaMKII<i>g</i>-siRNA+B, HKM transfected with CaMKII<i>g</i>-siRNA infected with <i>A. hydrophila</i>; HKM+CMZ+B, HKM pre-treated with CMZ infected with <i>A. hydrophila</i>; HKM+KN-93+B, HKM pre-treated with KN-93 infected with <i>A. hydrophila</i>; HKM+KN-92+B, HKM pre-treated with KN-92 infected with <i>A. hydrophila</i>; HKM+STO-609+B, HKM pre-treated with STO-609 infected with <i>A. hydrophila</i>; HKM+calpain-2<i>i</i>+B, HKM pre-treated with calpain-2<i>i</i> infected with <i>A. hydrophila.</i></p

List of degenerate primers.

<p>List of degenerate primers.</p

CaM-CaMKII-cAMP/PKA-ERK 1/2 axis leads to caspase-3 mediated apoptosis of <i>A. hydrophila</i>-infected HKM.

<p><i>A. hydrophila</i>-elevated Ca²⁺ leads to downstream calpain-2 activation and CaM expression. The pathways converge at CaMKII<i>g</i> to induce cAMP/PKA mediated activation of ERK 1/2 leading to caspase-3 mediated apoptosis of <i>A. hydrophila</i>-infected HKM.</p

<i>A. hydrophila</i> infection leads to increased CaM expression in HKM.

<p>(A) HKM transfected separately with CaM-siRNA, scrambled siRNA or pre-treated with CMZ, Vp, Nf, EGTA, BAPTA/AM for different time periods were infected with <i>A. hydrophila</i> and CaM protein content measured in the lysates 2 h p.i. using EIA kit. HKM pre-treated with CMZ or transfected with CaM-siRNA or scrambled siRNA were infected with <i>A. hydrophila</i> and checked for (B) Hoechst 33342 positive cells and caspase-3 activity and (C) AV-PI staining 24 h p.i. (D) HKM transfected with CaM-siRNA or scrambled siRNA were infected with <i>A. hydrophila</i> and CaM mRNA expression detected by real time PCR 2 h p.i. Vertical bars represent mean ± SE (n = 6). <b>*</b>P<0.05, compared to HKM; <b>^γ</b>P<0.05, compared to HKM+Sc; <b>^#</b>P<0.05, compared to HKM+B; <b>^±</b>P<0.05, compared to HKM+Sc+B. HKM, control head kidney macrophage; HKM+Sc, HKM transfected with scrambled siRNA; HKM+B, HKM infected with <i>A. hydrophila</i>; HKM+Sc+B, HKM transfected with scrambled siRNA infected with <i>A. hydrophila</i>; HKM+CaM-siRNA+B, HKM transfected with CaM-siRNA infected with <i>A. hydrophila</i>; HKM+BAPTA/AM+B, HKM pre-treated with BAPTA/AM infected with <i>A. hydrophila</i>; HKM+EGTA+B, HKM pre-treated with EGTA infected with <i>A. hydrophila</i>; HKM+Nf+B, HKM pre-treated with Nf infected with <i>A. hydrophila</i>; HKM+Vp+B, HKM pre-treated with Vp infected with <i>A. hydrophila</i>; HKM+CMZ+B, HKM pre-treated with CMZ infected with <i>A. hydrophila</i>.</p