HIF1 and ID1 Interplay Confers Adaptive Survival to HIF1α-Inhibition.

Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.

The AML microenvironment catalyzes a stepwise evolution to gilteritinib resistance.

Our study details the stepwise evolution of gilteritinib resistance in FLT3-mutated acute myeloid leukemia (AML). Early resistance is mediated by the bone marrow microenvironment, which protects residual leukemia cells. Over time, leukemia cells evolve intrinsic mechanisms of resistance, or late resistance. We mechanistically define both early and late resistance by integrating whole-exome sequencing, CRISPR-Cas9, metabolomics, proteomics, and pharmacologic approaches. Early resistant cells undergo metabolic reprogramming, grow more slowly, and are dependent upon Aurora kinase B (AURKB). Late resistant cells are characterized by expansion of pre-existing NRAS mutant subclones and continued metabolic reprogramming. Our model closely mirrors the timing and mutations of AML patients treated with gilteritinib. Pharmacological inhibition of AURKB resensitizes both early resistant cell cultures and primary leukemia cells from gilteritinib-treated AML patients. These findings support a combinatorial strategy to target early resistant AML cells with AURKB inhibitors and gilteritinib before the expansion of pre-existing resistance mutations occurs.

Discovery and Characterization of Recurrent, Targetable ALK Fusions in Leiomyosarcoma.

Soft-tissue sarcomas such as leiomyosarcoma pose a clinical challenge because systemic treatment options show only modest therapeutic benefit. Discovery and validation of targetable vulnerabilities is essential. To discover putative kinase fusions, we analyzed existing transcriptomic data from leiomyosarcoma clinical samples. Potentially oncogenic ALK rearrangements were confirmed by application of multiple RNA-sequencing fusion detection algorithms and FISH. We functionally validated the oncogenic potential and targetability of discovered kinase fusions through biochemical, cell-based (Ba/F3, NIH3T3, and murine smooth muscle cell) and in vivo tumor modeling approaches. We identified ALK rearrangements in 9 of 377 (2.4%) patients with leiomyosarcoma, including a novel KANK2-ALK fusion and a recurrent ACTG2-ALK fusion. Functional characterization of the novel ALK fusion, KANK2-ALK, demonstrates it is a dominant oncogene in Ba/F3 or NIH3T3 model systems, and has tumorigenic potential when introduced into smooth muscle cells. Oral monotherapy with targeted ALK kinase inhibitor lorlatinib significantly inhibits tumor growth and prolongs survival in a murine model of KANK2-ALK leiomyosarcoma. These results provide the first functional validation of a targetable oncogenic kinase fusion as a driver in a subset of leiomyosarcomas. Overall, these findings suggest that some soft-tissue sarcomas may harbor previously unknown kinase gene translocations, and their discovery may propel new therapeutic strategies in this treatment-refractory cancer. IMPLICATIONS: A subset of leiomyosarcomas harbor previously unrecognized oncogenic ALK fusions that are highly responsive to ALK inhibitors and thus these data emphasize the importance of detailed genomic investigations of leiomyosarcoma tumors.

Integration of genomic, transcriptomic and functional profiles of aggressive osteosarcomas across multiple species.

In complex, highly unstable genomes such as in osteosarcoma, targeting aberrant checkpoint processes (metabolic, cell cycle or immune) may prove more successful than targeting specific kinase or growth factor signaling pathways. Here, we establish a comparative oncology approach characterizing the most lethal osteosarcomas identified in a biorepository of tumors from three different species: human, mouse and canine. We describe the development of a genetically-engineered mouse model of osteosarcoma, establishment of primary cell cultures from fatal human tumors, and a biorepository of osteosarcoma surgical specimens from pet dogs. We analyzed the DNA mutations, differential RNA expression and in vitro drug sensitivity from two phenotypically-distinct cohorts: tumors with a highly aggressive biology resulting in death from rapidly progressive, refractory metastatic disease, and tumors with a non-aggressive, curable phenotype. We identified ARK5 (AMPK-Related Protein Kinase 5, also referred to as NUAK Family Kinase 1) as a novel metabolic target present in all species, and independent analyses confirmed glucose metabolism as the most significantly aberrant cellular signaling pathway in a model system for highly metastatic tumors. Pathway integration analysis identified Polo Like Kinase 1 (PLK1)-mediated checkpoint adaptation as critical to the survival of a distinctly aggressive osteosarcoma. The tumor-associated macrophage cytokine CCL18 (C-C Motif Chemokine Ligand 18) was significantly over-expressed in aggressive human osteosarcomas, and a clustering of mutations in the BAGE (B Melanoma Antigen) tumor antigen gene family was found. The theme of these features of high risk osteosarcoma is checkpoint adaptations, which may prove both prognostic and targetable.

HDAC4 protein regulates HIF1α protein lysine acetylation and cancer cell response to hypoxia.

Hypoxia-inducible factor 1 α (HIF1α) is an essential part of the HIF-1 transcriptional complex that regulates angiogenesis, cellular metabolism, and cancer development. In von Hippel-Lindau (VHL)-null kidney cancer cell lines, we reported previously that HIF1α proteins can be acetylated and inhibited by histone deacetylase (HDAC) inhibitors or specific siRNA against HDAC4. To investigate the mechanism and biological consequence of the inhibition, we have generated stable HDAC4 knockdown via shRNA in VHL-positive normal and cancer cell lines. We report that HDAC4 regulates HIF1α protein acetylation and stability. Specifically, the HIF1α protein acetylation can be increased by HDAC4 shRNA and decreased by HDAC4 overexpression. HDAC4 shRNA inhibits HIF1α protein stability. In contrast, HDAC1 or HDAC3 shRNA has no such inhibitory effect. Mutations of the first five lysine residues (lysine 10, 11, 12, 19, and 21) to arginine within the HIF1α N terminus reduce protein acetylation but render the mutant HIF1α protein resistant to HDAC4 and HDACi-mediated inhibition. Functionally, in VHL-positive cancer cell lines, stable inhibition of HDAC4 decreases both the HIF-1 transcriptional activity and a subset of HIF-1 hypoxia target gene expression. On the cellular level, HDAC4 inhibition reduces the hypoxia-related increase of glycolysis and resistance to docetaxel chemotherapy. Taken together, the novel biological relationship between HDAC4 and HIF1α presented here suggests a potential role for the deacetylase enzyme in regulating HIF-1 cancer cell response to hypoxia and presents a more specific molecular target of inhibition.

ID1 enhances docetaxel cytotoxicity in prostate cancer cells through inhibition of p21.

To identify potential mechanisms underlying prostate cancer chemotherapy response and resistance, we compared the gene expression profiles in high-risk human prostate cancer specimens before and after neoadjuvant chemotherapy and radical prostatectomy. Among the molecular signatures associated with chemotherapy, transcripts encoding inhibitor of DNA binding 1 (ID1) were significantly upregulated. The patient biochemical relapse status was monitored in a long-term follow-up. Patients with ID1 upregulation were found to be associated with longer relapse-free survival than patients without ID1 increase. This in vivo clinical association was mechanistically investigated. The chemotherapy-induced ID1 upregulation was recapitulated in the prostate cancer cell line LNCaP. Docetaxel dose-dependently induced ID1 transcription, which was mediated by ID1 promoter E-box chromatin modification and c-Myc binding. Stable ID1 overexpression in LNCaP increased cell proliferation, promoted G(1) cell cycle progression, and enhanced docetaxel-induced cytotoxicity. These changes were accompanied by a decrease in cellular mitochondria content, an increase in BCL2 phosphorylation at serine 70, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. In contrast, ID1 siRNA in the LNCaP and C42B cell lines reduced cell proliferation and decreased docetaxel-induced cytotoxicity by inhibiting cell death. ID1-mediated chemosensitivity enhancement was in part due to ID1 suppression of p21. Overexpression of p21 in LNCaP-ID1-overexpressing cells restored the p21 level and reversed ID1-enhanced chemosensitivity. These molecular data provide a mechanistic rationale for the observed in vivo clinical association between ID1 upregulation and relapse-free survival. Taken together, it shows that ID1 expression has a novel therapeutic role in prostate cancer chemotherapy and prognosis.

CCL2 is induced by chemotherapy and protects prostate cancer cells from docetaxel-induced cytotoxicity.

BACKGROUND: Metastatic prostate cancer is either inherently resistant to chemotherapy or rapidly acquires this phenotype after chemotherapy exposure. In this study, we identified a docetaxel-induced resistance mechanism centered on CCL2. METHODS: We compared the gene expression profiles in individual human prostate cancer specimens before and after exposure to chemotherapy collected from previously untreated patients who participated in a clinical trial of preoperative chemotherapy. Subsequently, we used the gain- and loss-of-function approach in vitro to identify a potential mechanism underlying chemotherapy resistance. RESULTS: Among the molecular signatures associated with treatment, several genes that regulate the inflammatory response and chemokine activity were upregulated including a significant increase in transcripts encoding the CC chemokine CCL2. Docetaxel increased CCL2 expression in prostate cancer cell lines in vitro. CCL2-specific siRNA inhibited LNCaP and LAPC4 cell proliferation and enhanced the growth inhibitory effect of low-dose docetaxel. In contrast, overexpression of CCL2 or recombinant CCL2 protein stimulated prostate cancer cell proliferation and rescued cells from docetaxel-induced cytotoxicity. This protective effect of CCL2 was associated with activation of the ERK/MAP kinase and PI3K/AKT, inhibition of docetaxel-induced Bcl2 phosphorylation at serine 70, phosphorylation of Bad, and activation of caspase-3. The addition of a PI3K/AKT inhibitor Ly294002 reversed the CCL2 protection and was additive to docetaxel-induced toxicity. CONCLUSION: These results support a mechanism of chemotherapy resistance mediated by cellular stress responses involving the induction of CCL2 expression and suggest that inhibiting CCL2 activity could enhance therapeutic responses to taxane-based therapy.

The iroquois homeobox gene 5 is regulated by 1,25-dihydroxyvitamin D3 in human prostate cancer and regulates apoptosis and the cell cycle in LNCaP prostate cancer cells.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the most active metabolite of vitamin D3, has significant antitumor activity in a broad range of preclinical models of cancer. In this study, we show that the Iroquois homeobox gene 5 (Irx5) is down-regulated by 1,25(OH)2D3 in human prostate cancer samples from patients randomly assigned to receive weekly high-dose 1,25(OH)2D3 or placebo before radical prostatectomy. Down-regulation of Irx5 by 1,25(OH)2D3 was also shown in the human androgen-sensitive prostate cancer cell line LNCaP and in estrogen-sensitive MCF-7 breast cancer cells. Knockdown of Irx5 by RNA interference showed a significant reduction in LNCaP cell viability, which was accompanied by an increase in p21 protein expression, G2-M arrest, and an increase in apoptosis. The induced apoptosis was partially mediated by p53, and p53 protein expression was increased as a result of Irx5 knockdown. Cell survival was similarly reduced by Irx5 knockdown in the colon cancer cell line HCT 116 and in MCF-7 breast cancer cells, each being derived from clinical tumor types that seem to be inhibited by 1,25(OH)2D3. Overexpression of Irx5 led to a reduction of p21 and p53 expression. This is the first report that Irx5 is regulated by 1,25(OH)2D3 in humans and the first report to show that Irx5 is involved in the regulation of both the cell cycle and apoptosis in human prostate cancer cells. Irx5 may be a promising new therapeutic target in cancer treatment.