BackgroundCancer treatment with a variety of chemotherapeutic agents often is associated with delayed adverse neurological consequences. Despite their clinical importance, almost nothing is known about the basis for such effects. It is not even known whether the occurrence of delayed adverse effects requires exposure to multiple chemotherapeutic agents, the presence of both chemotherapeutic agents and the body's own response to cancer, prolonged damage to the blood-brain barrier, inflammation or other such changes. Nor are there any animal models that could enable the study of this important problem.ResultsWe found that clinically relevant concentrations of 5-fluorouracil (5-FU; a widely used chemotherapeutic agent) were toxic for both central nervous system (CNS) progenitor cells and non-dividing oligodendrocytes in vitro and in vivo. Short-term systemic administration of 5-FU caused both acute CNS damage and a syndrome of progressively worsening delayed damage to myelinated tracts of the CNS associated with altered transcriptional regulation in oligodendrocytes and extensive myelin pathology. Functional analysis also provided the first demonstration of delayed effects of chemotherapy on the latency of impulse conduction in the auditory system, offering the possibility of non-invasive analysis of myelin damage associated with cancer treatment.ConclusionsOur studies demonstrate that systemic treatment with a single chemotherapeutic agent, 5-FU, is sufficient to cause a syndrome of delayed CNS damage and provide the first animal model of delayed damage to white-matter tracts of individuals treated with systemic chemotherapy. Unlike that caused by local irradiation, the degeneration caused by 5-FU treatment did not correlate with either chronic inflammation or extensive vascular damage and appears to represent a new class of delayed degenerative damage in the CNS.

Background: Chemotherapy in cancer patients can be associated with serious short-and long-term adverse neurological effects, such as leukoencephalopathy and cognitive impairment, even when therapy is delivered systemically. The underlying cellular basis for these adverse effects is poorly understood.

Background The Wnt/β-catenin pathway is closely associated with osteosarcoma (OS) development and metastatic progression. We investigated the antitumor activity of Tegavivint, a novel β-catenin/transducin β-like protein 1 (TBL1) inhibitor, against OS employing in vitro, ex vivo, and in vivo cell line and patient-derived xenograft (PDX) models that recapitulate high risk disease. Methods The antitumor efficacy of Tegavivint was evaluated in vitro using established OS and PDX-derived cell lines. Use of an ex vivo three-dimensional pulmonary metastasis assay assessed targeting of β-catenin activity during micro- and macrometastatic development. The in vivo activity of Tegavivint was evaluated using chemoresistant and metastatic OS PDX models. Gene and protein expression were quantified by quantitative Reverse transcription polymerase chain reaction or immunoblot analysis. Bone integrity was determined via microCT. All statistical tests were two-sided. Results Tegavivint exhibited antiproliferative activity against OS cells in vitro and actively reduced micro- and macrometastatic development ex vivo. Multiple OS PDX tumors (n = 3), including paired patient primary and lung metastatic tumors with inherent chemoresistance, were suppressed by Tegavivint in vivo. We identified that metastatic lung OS cell lines (n = 2) exhibited increased stem cell signatures, including enhanced concomitant aldehyde dehydrogenase (ALDH1) and β-catenin expression and downstream activity, which were suppressed by Tegavivint (ALDH1: control group, mean relative mRNA expression = 1.00, 95% confidence interval [CI] = 0.68 to 1.22 vs Tegavivint group, mean = 0.011, 95% CI = 0.0012 to 0.056, P < .001; β-catenin: control group, mean relative mRNA expression = 1.00, 95% CI = 0.71 to 1.36 vs Tegavivint group, mean = 0.45, 95% CI = 0.36 to 0.52, P < .001). ALDH1high PDX-derived lung OS cells, which demonstrated enhanced metastatic potential compared with ALDHlow cells in vivo, were sensitive to Tegavivint. Toxicity studies revealed decreased bone density in male Tegavivint-treated mice (n = 4 mice per group). Conclusions Tegavivint is a promising therapeutic agent for advanced stages of OS via its targeting of the β-catenin/ALDH1 axis.

SP‐2577(Seclidemstat), an inhibitor of lysine‐specific demthylase KDM1A (LSD1) that is overexpressed in pediatric sarcomas, was evaluated against pediatric sarcoma xenografts. SP‐2577 (100 mg/kg/day × 28 days) statistically significantly (p < .05) inhibited growth of three of eight Ewing sarcoma (EwS), four of five rhabdomyosarcoma (RMS), and four of six osteosarcoma (OS) xenografts. The increase in EFS T/C was modest (<1.5) for all models except RMS Rh10 (EFS T/C = 2.8). There were no tumor regressions or consistent changes in dimethyl histone H3(K4), HOXM1, DAX1, c‐MYC and N‐MYC, or tumor histology/differentiation. SP‐2577 has limited activity against these pediatric sarcoma models at the dose and schedule evaluated.

There is a clear need to develop novel therapies that would overcome differentiation block and eliminate AML stem/progenitor cells. Genetic and epigenetic dysregulation of enhancers regulates expressions of myeloid lineage transcriptional regulators and their target genes in AML stem/progenitor cells. LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2), which regulates enhancer maintenance and transcription in AML stem/progenitor cells (LSCs). LSD1 is part of the repressor complexes involving HDACs, CoREST or GFI1 that mediate transcriptional repression and differentiation block in AML blast progenitor cells (BPCs). We had previously reported that treatment with the reversible LSD1 inhibitor (LSDi) SP2509 increases the permissive H3K4Me3 mark on the chromatin, associated with induction of p21, p27 and CEBPα levels, as well as of differentiation and loss of viability of AML BPCs (Leukemia. 2014; 28: 2155-64). In the present studies, we further evaluated the anti-AML efficacy of LSD1i-based combination with BET protein inhibitor (BETi). First, we determined that tet-inducible shRNA to KDM1A depleted protein levels of KDM1A, repressed c-Myc, but de-repressed p21, CD11b (ITGAM), CD86 and CEBPα, thereby inhibiting colony growth and modestly inducing lethality in genetically diverse cultured AML cell lines. Following sgRNA-directed, CRISPR/Cas9-mediated gene-editing of LSD1 in AML BPCs, surviving clones exhibited ~50% KDM1A levels and decreased c-Myc and DNMT1 expressions compared to the control AML BPCs. Treatment with either the reversible LSDi, SP2577 (Salarius Pharma), or with the irreversible LSDi ORY-1001, disrupted binding of KDM1A with CoREST. Following LSDi treatment, ATAC-Seq analyses demonstrated significant increase in the accessible chromatin of AML BPCs (represented by gained peaks). Gained ATAC-Seq peaks also involved the chromatin of MED11/13, LY96, CEBPB, RARA, CDKN1C and CD86 genes. ChIP-Seq analysis also showed increased H3K27Ac peaks in the chromatin of CD86, ITGAM, SAMHD1, TET2, MED12 and E2F1, and a reduction of peaks in RUNX1, CDK6, KIT, CTNNB1, HOXB5, FLT3 and MEIS1. RNA-Seq analyses after LSD1i treatment also showed significant perturbations (log2 fold-change >1.25 and p<0.05) in the mRNA expressions, including those of ITGAM, LY96, CD86, SAMHD1, IRF8, APAF1, CDK6, and KIT. Gene set enrichment analysis against Hallmark and Transcription Factor-Target datasets showed positive enrichment of E2F and GFI1 targets, as well as of IL2-STAT5 and TGF-beta signaling, but significant depletion (FDR q-values <0.1) of MYC-targets and genes involved in oxidative phosphorylation. QPCR and Western analyses following LSD1i treatment confirmed significant up regulation of mRNA and protein levels, respectively, of ITGAM, CD86 and LY96 in cultured and primary patient-derived AML BPCs. This was associated with morphologic features of differentiation and inhibition of colony growth in AML cells (OCI-AML5, MOLM13, THP1 and MV4-11) (p < 0.01). We also queried for expression mimickers (EMs) through connectivity mapping of the mRNA signature following LSD1i treatment, utilizing the LINCS1000-CMap analyses. Among the top EM hits were BET protein inhibitors (BETis). Treatment with BETi or BET protein degraders (PROTACs) depleted LSD1 levels in AML BPCs. Utilizing ChIP-Seq data, we also noted that LSD1 promoter is occupied by BET protein BRD4 in the AML cells. Consistent with this, treatment with the BETi OTX015 depleted KDM1A expression in AML cells. Notably, co-treatment with LSDi (SP2577 or ORY-1001) and OTX015 induced synergistic lethality in AML BPCs, including of CD34+, CD38-, Lin- AML stem/progenitor cells (combination indices < 1.0). This was associated with greater depletion of c-Myc, c-Myb and PU.1, but greater induction of p21 and p27. LSD1i or BETi treatment significantly improved survival of the immune-depleted mice (compared to the control mice) engrafted with the AML OCI-AML5 cells or patient-derived xenograft (PDX) models of AML (p < 0.01). Collectively, these findings elucidate the molecular mechanisms and strongly support further in vivo testing and pre-clinical development of LSD1i-based combinations with BETi against AML BPCs. Disclosures Soldi: Beta Cat Pharma: Employment. Han:Beta Cat Pharma: Employment. DiNardo:Agios: Consultancy, Other: Advisory role; Bayer: Other: Advisory role; Celgene: Other: Advisory role; Medimmune: Other: Advisory role; Karyopharm: Other: Advisory role; AbbVie: Consultancy, Other: Advisory role. Kadia:Celgene: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Abbvie: Consultancy; Abbvie: Consultancy; BMS: Research Funding; Jazz: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Research Funding; Novartis: Consultancy. Khoury:Stemline Therapeutics: Research Funding.