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 (>90% viability at 48 h), good (>50%), or unusable (<50%). 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.

Reduced levels of Hspa9 attenuate Stat5 activation in mouse B cells.

HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndrome (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. Although homozygous knockout of Hspa9 is embryonically lethal, mice with heterozygous deletion of Hspa9 (Hspa9(+/-)) are viable and have a 50% reduction in Hspa9 expression. Hspa9(+/-) mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9(+/-) mice have a cell-intrinsic reduction in bone marrow colony-forming unit-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9(+/-) B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNA interference and observed reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B lymphopoiesis in vivo. Knockdown of Hspa9 in an interleukin 7 (IL-7)-dependent mouse B-cell line reduced signal transducer and activator of transcription 5 (Stat5) phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B cells. Collectively, these data imply a role for Hspa9 in B lymphopoiesis and Stat5 activation downstream of IL-7 signaling.

Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice.

Heterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when p53 is mutated. Mutagenesis screens in zebrafish and mice identified Hspa9 as a del(5q31.2) candidate gene that may contribute to MDS and AML pathogenesis, respectively. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, we knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow-transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors, but not myeloid or megakaryocytic precursors, and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors c-kit(+)/lineage(-)/Sca-1(+) (KLS) and megakaryocyte/erythrocyte progenitor (MEP) were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.