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Colorectal cancer (CRC) incidence is rising globally. Hence, preventing this disease is a high priority. With this aim, we determined the CRC prevention potential of the TRAIL-inducing small molecule ONC201/TIC10 using a preclinical model representing high-risk familial adenomatous polyposis (FAP) patients, Apc min/+ mice. Prior to the efficacy study, optimal and non-toxic doses of ONC201 were determined by testing five different doses of ONC201 (0-100 mg/kg body weight (BW); twice weekly by oral gavage) in C57BL/6J mice ( n =6/group) for 6 weeks. BW gain, organ weights and histopathology, blood profiling, and the plasma liver enzyme profile suggested no toxicities of ONC201 at doses up to 100 mg/kg BW. For efficacy determination, beginning at six weeks of age, groups of Apc min/+ male and female mice ( n ≥20) treated with colon carcinogen azoxymethane (AOM) (AOM- Apc min/+ ) were administered ONC201 (0, 25, and 50 mg/kg BW) as above up to 20 weeks of age. At termination, efficacy was determined by comparing the incidence and multiplicity of intestinal tumors between vehicle- and drug-treated groups. ONC201 showed a strong suppressive effect against the development of both large and small intestinal tumors in male and female mice. Apc min/+ mice treated with ONC201 (50 mg/kg BW) showed >50% less colonic tumor incidence ( P <0.0002) than controls. Colonic tumor multiplicity was also significantly reduced by 68% in male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in controls; P <0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in controls; P <0.0003) with ONC201 treatment (50 mg/kg BW). Small intestinal polyps were reduced by 68% in male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in controls; P <0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in controls; P <0.0001). Molecular analysis of the tumors suggested an increase in TRAIL, DR5, cleaved caspases 3/7/8, Fas-associated death domain protein (FADD), and p21 (WAF1) in response to drug treatment. Serum analysis indicated a decrease in pro-inflammatory serum biomarkers, such as IL1β, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated mice compared with controls. Our data demonstrated excellent chemopreventive potential of orally administered ONC201 against intestinal tumorigenesis in the AOM- Apc min/+ mouse model., Competing Interests: W.S.E-D. is a co-founder of Oncoceutics, Inc., a subsidiary of Chimerix. Dr. El-Deiry has disclosed his relationship with Oncoceutics and potential conflict of interest to his academic institution/employer and is fully compliant with NIH and institutional policy that is managing this potential conflict of interest. Other authors have no potential conflicts of interest., (AJCR Copyright © 2022.)

Hydroxyurea (HU) has been widely used in sickle cell disease. Its potential long-term risk for carcinogenesis or leukemogenic risk remains undefined. Here, we report a 26 y old African-American female with Sickle Cell Disease (SCD) who developed refractory/relapsed acute myeloid leukemia (AML) 6 months after 26 months of HU use. That patient's cytogenetics and molecular genetics analyses demonstrated a complex mutation profile with 5q deletion, trisomy 8, and P53 deletion (deletion of 17p13.1). P53 gene sequence studies revealed a multitude of somatic mutations that most suggest a treatment-related etiology. The above-mentioned data indicates that the patient may have developed acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) as a direct result of HU exposure.

Nucleotide metabolism in cancer cells can influence malignant behavior and intrinsic resistance to therapy. Here we describe p53-dependent control of the rate-limiting enzyme in the pyrimidine catabolic pathway, dihydropyrimidine dehydrogenase (DPYD) and its effect on pharmacokinetics of and response to 5-fluorouracil (5-FU). Using in silico/chromatin-immunoprecipitation (ChIP) analysis we identify a conserved p53 DNA-binding site (p53BS) downstream of the DPYD gene with increased p53 occupancy following 5-FU treatment of cells. Consequently, decrease in Histone H3K9AC and increase in H3K27me3 marks at the DPYD promoter are observed concomitantly with reduced expression of DPYD mRNA and protein in a p53-dependent manner. Mechanistic studies reveal inhibition of DPYD expression by p53 is augmented following thymidylate synthase (TS) inhibition and DPYD repression by p53 is dependent on DNA-dependent protein kinase (DNA-PK) and Ataxia telangiectasia mutated (ATM) signaling. In-vivo, liver specific Tp53 loss increases the conversion of 5-FU to 5-FUH 2 in plasma and elicits a diminished 5-FU therapeutic response in a syngeneic colorectal tumor model consistent with increased DPYD-activity. Our data suggest that p53 plays an important role in controlling pyrimidine catabolism through repression of DPYD expression, following metabolic stress imposed by nucleotide imbalance. These findings have implications for the toxicity and efficacy of the cancer therapeutic 5-FU.

We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patient's peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo . Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses., Competing Interests: CONFLICTS OF INTEREST L.G. and N.C.N. are employees of Morphotek Inc. The remaining authors declare that they have no conflict of interest.

The release of inflammatory cytokines has been implicated in the toxicity of conventional radiotherapy (CRT). Transforming growth factor β (TGF-β) has been suggested to be a risk marker for pulmonary toxicity following radiotherapy. Pulsed low-dose rate radiotherapy (PLDR) is a technique that involves spreading out a conventional radiotherapy dose into short pulses of dose with breaks in between to reduce toxicities. We hypothesized that the more tolerable toxicity profile of PLDR compared with CRT may be related to differential expression of inflammatory cytokines such as TGF-β in normal tissues. To address this, we analyzed tissues from mice that had been subjected to lethal doses of CRT and PLDR by histology and immunohistochemistry (IHC). Equivalent physical doses of CRT triggered more cellular atrophy in the bone marrow, intestine, and pancreas when compared with PLDR as indicated by hematoxylin and eosin staining. IHC data indicates that TGF-β expression is increased in the bone marrow, intestine, and lungs of mice subjected to CRT as compared with tissues from mice subjected to PLDR. Our in vivo data suggest that differential expression of inflammatory cytokines such as TGF-β may play a role in the more favorable normal tissue late response following treatment with PLDR.

Previous studies have linked increased frequency of glycosylphosphatidylinositol-anchor protein (GPI-AP) deficiency with genomic instability and the risk of carcinogenesis. However, the underlying mechanism is still not clear. A randomForest analysis of the gene expression array data from 55 MDS patients (GSE4619) demonstrated a significant (p = 0.0007) correlation (Pearson r =-0.4068) between GPI-anchor biosynthesis gene expression and genomic instability, in which PIGN, a gene participating in GPI-AP biosynthesis, was ranked as the third most important in predicting risk of MDS progression. Furthermore, we observed that PIGN gene expression aberrations (increased transcriptional activity but diminished to no protein production) were associated with increased frequency of GPI-AP deficiency in leukemic cells during leukemic transformation/progression. PIGN gene expression aberrations were attributed to partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination which were confirmed by examining the RNA-seq data from a group of AML patients (phs001027.v1.p1). PIGN gene expression aberration correlated with the elevation of genomic instability marker expression that was independent of the TP53 regulatory pathway. Suppression/elimination of PIGN protein expression caused a similar pattern of genomic instability that was rescued by PIGN restoration. Finally, we found that PIGN bound to the spindle assembly checkpoint protein, MAD1, and regulated its expression during the cell cycle. In conclusion, PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevents leukemic transformation/progression.

Purpose and Experimental Design: Anaplastic thyroid cancer (ATC) comprises approximately 2% of all thyroid cancers, and its median survival rate remains poor. It is responsible for more than one third of thyroid cancer-related deaths. ATC is frequently resistant to conventional therapy, and NFκB signaling has been proposed to be a feature of the disease. We aimed to assess the activity of the antimalaria drug quinacrine known to target NFκB signaling in combination with the clinically relevant kinase inhibitor sorafenib in ATC cells. The presence of NFκB-p65/RELA and its target MCL1 was demonstrated in ATC by meta-data gene set enrichment analysis and IHC. We assessed the responses of a panel of human ATC cell lines to quinacrine and sorafenib in vitro and in vivo RESULTS: We detected increased expression of NFκB-p65/RELA and MCL1 in the nucleus of a subset of ATC compared with non-neoplastic thyroid. ATC cells were found to respond with additive/synergistic tumor cell killing to the combination of sorafenib plus quinacrine in vitro, and the drug combination improves survival of immunodeficient mice injected orthotopically with ATC cells as compared with mice administered either compound alone or doxorubicin. We also demonstrate that the combination of sorafenib and quinacrine is well tolerated in mice. At the molecular level, quinacrine and sorafenib inhibited expression of prosurvival MCL1, pSTAT3, and dampened NFκB signaling., Conclusions: The combination of quinacrine and sorafenib targets emerging molecular hallmarks of ATC and shows promising results in clinically relevant models for the disease. Further testing of sorafenib plus quinacrine can be conducted in ATC patients. Clin Cancer Res; 22(24); 6192-203. ©2016 AACR., (©2016 American Association for Cancer Research.)

The DNA damage response (DDR) gene cell cycle checkpoint kinase 2 (Chk2) triggers programmed cell death and lethal radiation-induced toxicity in mice in vivo. However, it is not well established to what extent targeting of Chk2 may protect from dose-limiting toxicities (DLT) inflicted by mainstay cancer chemotherapy. We screened different classes of chemotherapy in wild type and Chk2-deficient cells. Here we show that loss of Chk2 protect from cell death in vitro and lethal toxicity in vivo following treatment with topoisomerase II (TOP2)-inhibitors whereas no such protection was observed following treatment with topoisomerase I (TOP1) inhibitors. Furthermore, through combined in silico and functional screens of the Diversity Set II (NCI/NTP) chemical library we identified the carbanilide-derivative NSC105171, also known as ptu-23, as a novel Chk2 inhibitor (Chk2i). Indeed, NSC105171 can be administered safely to mice to countermeasure etoposide-induced toxicity. Incorporation of Chk2i into chemotherapy protocols employing TOP2-inhibitors may be an effective strategy to prevent DLT's without interfering with treatment., Competing Interests: The authors declare that they have no conflicting interests.

The combination of TRAIL death receptor agonists and radiochemotherapy to treat advanced cancers continues to be investigated in clinical trials. We previously showed that normal cells with a functional DNA damage response (DDR) upregulate the expression of death-inducing receptor DR5/TRAILR2/TNFRSF10B in a p53-dependent manner that sensitizes them to treatment with DR5 agonists. However, it is unclear if targeting DR5 selectively sensitizes cancer cells to agonist treatment following exposure to DNA-damaging chemotherapy, and to what extent normal tissues are targeted. Here, we show that the combined administration of the DR5 agonistic monoclonal antibody (mAb) and chemotherapy to wild-type mice triggered synergistic gastrointestinal toxicities (GIT) that were associated with the death of Lgr5(+) crypt base columnar stem cells in a p53- and DR5-dependent manner. Furthermore, we confirmed that normal human epithelial cells treated with the human DR5-agonistic mAb and chemotherapeutic agents were also greatly sensitized to cell death. Interestingly, our data also indicated that genetic or pharmacologic targeting of Chk2 may counteract GIT without negatively affecting the antitumor responses of combined DR5 agonist/chemotherapy treatment, further linking the DDR to TRAIL death receptor signaling in normal cells. In conclusion, the combination of DR5-targeting agonistic mAbs with DNA damaging chemotherapy may pose a risk of developing toxicity-induced conditions, and the effects of mAb-based strategies on the dose-limiting toxicity of chemotherapy must be considered when establishing new combination therapies., Competing Interests: The authors have no conflicts to disclose, (©2015 American Association for Cancer Research.)

Introduction: While apoptosis is critical for maintaining homeostasis in normal cells, defective apoptosis contributes to the survival of cancer cells. TNF-related apoptosis-inducing ligand (TRAIL)-targeted therapy has attracted significant effort for treating cancer, but the clinical results have revealed limitations. The authors review the current status of development of TRAIL-targeted therapy with an outlook towards the future., Areas Covered: Recombinant human proteins, small molecules and agonistic monoclonal antibodies targeting death receptors that trigger TRAIL-mediated apoptosis are covered in this article. The authors review both intrinsic and extrinsic apoptotic pathways, highlighting how the apoptosis serves as a promising therapeutic target. They also review different categories of TRAIL pathway targeting agents and provide a brief overview of clinical trials using these agents. The authors discuss the limitations of conventional approaches for targeting the TRAIL pathway as well as future directions., Expert Opinion: The development of better combination partners for pro-apoptotic TRAIL pathway modulators including novel agents inhibiting anti-apoptotic molecules or targeting alternative resistance pathways may improve the chances for anti-tumor responses in the clinic. Developing predictive biomarkers via circulating tumor cells/DNA, apoptosis signal products, and genetic signatures/protein biomarkers from tumor tissue are also suggested as future directions.

Approximately 1.4 million people of the US population suffer from Inflammatory Bowel Disease (IBD) of which the most common conditions are ulcerative colitis (UC) and Crohn disease (CD). Colonoscopy and small bowel follow through are considered the current gold standard in diagnosing IBD. However, improved imaging and increased diagnostic sensitivity could be beneficial. Optical molecular imaging has the potential to become a powerful and practical tool for early detection, image-guided biopsy, and surgery in diagnosing and treating patients with IBD. Here we used a well characterized chemical model to initiate experimental IBD in mice by feeding with dextran sulfate sodium (DSS) containing drinking water in an attempt to investigate the utility of non-invasive infrared (NIR) optical imaging in the detection gastrointestinal (GI) injury. We employed a "smart probe" (ProSense680) cleaved and fluorescently activated in the NIR-spectrum by various forms of secreted cathepsins. This probe has previously been shown to serve as a biomarker for the homing of inflammatory cells to injury. Our investigation suggests that NIR optical imaging can detect cathepsin-dependent probe cleavage non-invasively in animals with DSS-induced IBD. Increased tissue probe-retention and fluorescence was associated with increased infiltration of inflammatory cells, epithelial atrophy and sterilization of the mucosa. Furthermore, using NIR-imaging ex vivo we were able to document regional "hot spots" of inflammatory damage to the large intestine suggesting this method potentially could be coupled with colonoscopy investigation to aid in the sampling and the diagnostics of IBD.

Bone marrow-derived cells (BMDCs) participate in the growth and spread of tumors of the breast, brain, lung, and stomach. To date, there are limited reports of bone marrow involvement in colon cancer pathogenesis, but such findings would have the potential to generate novel treatments for colon cancer patients. We have established a mouse model for imaging BMDCs from whole tumor to single-cell resolution, whereby the bone marrow of lethally irradiated host animals is reconstituted with EGFP-expressing bone marrow cells from matched TgActb(EGFP) donors. The BM transplants yield mice with fluorescently labeled bone marrow, and so BMDCs can subsequently be monitored within a tumor through optical imaging. Successful BM reconstitution was confirmed at 8 weeks after transplantation, when surviving BALB/c mice were injected with CT26 mouse colon cancer cells. We find that up to 45% of cells dissociated from the tumors are GFP(+) and approximately 50% of Lin(+), CD11b(+), and CD3(+) cells express high levels of GFP. Notably, tumor growth is reduced in BM transplanted animals, compared with untransplanted host mice or EGFP-expressing BM donor mice. A needed next step is to separate the molecular and cellular (eg, T cells, NK cells, macrophages) bases of the antitumor effect of the BMDCs from any protumorigenic effect that could be subverted for therapeutic gain., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Efforts to improve efficacy and minimize toxicity have led to pharmacokinetic monitoring of plasma 5-Fluorouracil (5-FU) levels in colorectal cancer patients undergoing chemotherapy. We observed variation in basal 5-FU levels in 21 patients and significant variation during subsequent dose optimization. Tumor KRAS, BRAF mutations and TS mRNA levels were determined. Regimens included FOLFOX6 + Avastin (N = 8), FOLFOX6 (N = 11), FOLFIRI (N = 1) and FOLFOX4 (N = 1). Mutations identified in tumors included G12V KRAS (N = 2), G12A KRAS (N = 1), and V600E BRAF (N = 3). Six-of-eleven patients with normalized tumor TS mRNA levels < 4.0 had a 5-FU AUC of 20 mg.h/L or greater, and 80% of patients (4 of 5) with TS levels > 4.0 had a plasma 5-FU AUC of less than or equal to 20 mg.h/L. Approximately 2/3 of patients achieved therapeutic 5-FU AUC levels with 0-2 dose adjustments while a sub-group of ~1/3 of patients slowly achieved therapeutic levels (> 3-4 dose increases leading to supra-therapeutic 5-FU and subsequent reductions to lesser than original doses). Liver metastases and tumor TS levels did not fully account for variable 5-FU AUC optimization patterns. The 5-FU level during continuous infusion was half-therapeutic in one patient who received FOLFOX4. The observed heterogeneous patterns at baseline and during dose optimization of 5-FU levels suggest variations in 5-FU metabolism among treated patients. Physiological and/or genetic differences underlying heterogeneity in 5-FU levels during dose optimization require further study of patient demographics, single nucleotide polymorphisms in Dihydropyrimidine Dehydrogenase (DPD), TS, or other genes that impact 5-FU metabolism and gene expression changes in liver after 5-FU therapy.