Genetic Analysis of Biopsy Tissues from Colorectal Tumors in Patients with Ulcerative Colitis.

BACKGROUND/OBJECTIVES: Colorectal neoplasia developing from ulcerative colitis mucosa (CRNUC) can be divided into ulcerative colitis-associated neoplasia (UCAN) and non-UCAN; however, it is often difficult to distinguish UCAN from non-UCAN during a biopsy diagnosis. We investigated whether a genomic analysis could improve the diagnostic accuracy of UCAN using biopsy specimens. METHODS: In step 1, 14 CRNUCs were used to examine whether the genomic landscape of biopsy and resection specimens matched. In step 2, we investigated the relationship between the genomic landscapes and the pathological diagnosis of 26 CRNUCs. The cancer genome was analyzed by deep sequencing using a custom panel of 27 genes found to be mutated in our previous CRNUC analysis. RESULTS: In step 1, of the 27 candidate genes, 14 were mutated. The concordance rate of the pathogenic mutations in these 14 genes between the biopsy and resection specimens was 29% (4/14), while that of the pathogenic mutations in TP53 and KRAS was 79% (11/14). In step 2, the pathological diagnosis of biopsy specimens using only hematoxylin and eosin (HE) staining had a sensitivity of 33% and an accuracy of 38% for UCAN diagnosis. On the other hand, the combination of the HE pathology and p53 immunohistochemical staining had a sensitivity of 73% and an accuracy of 85% for UCAN diagnosis, while the combination of HE staining and a TP53 mutation had a sensitivity of 87% and an accuracy of 88% for UCAN diagnosis. CONCLUSIONS: An evaluation of TP53 mutations in biopsy specimens may be useful for diagnosing UCAN. However, further studies with larger sample sizes are required before this can be applied in clinical practice.

WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations.

Inhibition of WEE1, a key regulator of the G2/M checkpoint of the cell cycle, induces apoptosis by initiating mitosis without repairing DNA damage. However, the effects of WEE1 inhibitors on the tumor immune microenvironment in colorectal cancer (CRC) remain unclear. Here, we investigated the association between WEE1 expression and CRC clinicopathological features using surgically resected CRC specimens and assessed the antitumor effects of a WEE1 inhibitor using CRC cell lines and orthotopic transplantation mouse models. WEE1 expression was not correlated with the clinicopathological features of CRC. The WEE1 inhibitor suppressed cell proliferation in a concentration-dependent manner in all CRC cell lines. It also increased the percentage of cells in the G2/M phase and apoptotic cells, especially in cell lines with p53 mutations, but did not alter these cell percentages in most p53 wild-type cell lines. In the orthotopic mouse model of CRC, tumor volume was significantly reduced in the WEE1 inhibitor-treated group compared to that in the control group. RNA sequencing and immunohistochemistry analyses of mouse tumors revealed that treatment with the WEE1 inhibitor activated tumor immunity and suppressed stromal reactions. These results demonstrate the potential antitumor effects of WEE1 inhibitors in CRC, particularly in patients with p53 mutations.

Pexidartinib and Immune Checkpoint Inhibitors Combine to Activate Tumor Immunity in a Murine Colorectal Cancer Model by Depleting M2 Macrophages Differentiated by Cancer-Associated Fibroblasts.

Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are known to play supportive roles in tumor development and progression, but their interactions in colorectal cancer (CRC) remain unclear. Here, we investigated the effects of colon-cancer-derived CAFs on TAM differentiation, migration, and tumor immunity, both in vitro and in vivo. When co-cultured with monocytes, CAFs attracted monocytes and induced their differentiation into M2 macrophages. Immunohistology of surgically resected human CRC specimens and orthotopically transplanted mouse tumors revealed a correlation between numbers of CAFs and numbers of M2 macrophages. In a mouse model of CRC orthotopic transplantation, treatment with an inhibitor of the colony-stimulating factor-1 receptor (PLX3397) depleted M2 macrophages and increased CD8-positive T cells infiltrating the tumor nest. While this treatment had a minor effect on tumor growth, combining PLX3397 with anti-PD-1 antibody significantly reduced tumor growth. RNA-seq following combination therapy showed activation of tumor immunity. In summary, CAFs are involved in the induction and mobilization of M2 macrophage differentiation in the CRC tumor immune microenvironment, and the combination of cancer immunotherapy and PLX3397 may represent a novel therapeutic option for CRC.

Analysis of anti-tumor effect and mechanism of GLS1 inhibitor CB-839 in colorectal cancer using a stroma-abundant tumor model.

BACKGROUND: Glutaminase 1 (GLS1), a key enzyme in glutamine metabolism in cancer cells, acts as a tumor promoter and could be a potential therapeutic target. CB-839, a GLS1-specific inhibitor, was developed recently. Herein, we aimed to elucidate the anti-tumor effects and mechanism of action of CB-839 in colorectal cancer (CRC). METHODS: Using the UCSC Xena public database, we evaluated GLS1 expression in various cancers. Immunostaining for GLS1 was performed on 154 surgically resected human CRC specimens. Subsequently, we examined the GLS1 mRNA expression levels in eight CRC cell lines and evaluated the association between GLS1 expression and CB-839 efficacy. To create a reproducible CRC model with abundant stroma and an allogeneic immune response, we co-transplanted CT26 and stem cells into BALB/c mice and treated them with CB-839. Finally, RNA sequencing of mouse tumors was performed. RESULTS: Database analysis showed higher GLS1 expression in CRC tissues than in normal colon tissues. Clinical samples from 114 of the 154 patients with CRC showed positive GLS1 expression. GLS1 expression in clinical CRC tissues correlated with vascular invasion. CB-839 treatment inhibited cancer cell proliferation depending on GLS1 expression in vitro and inhibited tumor growth and metastasis in the CRC mouse model. RNA sequencing revealed that CB-839 treatment inhibited stromal activation, tumor growth, migration, and angiogenesis. These findings were validated through in vitro and in vivo experiments and clinical specimen analysis. CONCLUSIONS: GLS1 expression in CRC plays important roles in tumor progression. CB-839 has inhibitory effects on cancer proliferation and the tumor microenvironment.

Synchronous gastric MALT lymphoma and gastric adenocarcinoma of fundic gland type arising from a hamartomatous inverted polyp in a Helicobacter pylori naive patient.

We present a rare case that showed the coexistence of gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma in Helicobacter pylori-naive stomach. A 72-year-old man was followed up after surgery for epithelial carcinoma of the glottis at the Department of Otolaryngology. He underwent an upper gastrointestinal endoscopy for an abnormal PET-CT accumulation, which revealed gastric adenocarcinoma of fundic gland type in the gastric fundus and MALT lymphoma in the upper gastric body. Hence, we performed an endoscopic submucosal dissection for gastric cancer and diagnosed gastric adenocarcinoma of fundic gland type derived from a hamartomatous-inverted polyp. Subsequently, Gastric MALT lymphoma was treated with radiation therapy because the API2-MALT1 gene was positive and the Helicobacter pylori infection was negative. A complete response was observed. Even in Hp-naive stomachs, cases such as the present case are complicated by special types of gastric cancer and MALT lymphoma, and endoscopic examination should be performed with these diseases in mind.

The Anti-Tumor Effect of the Newly Developed LAT1 Inhibitor JPH203 in Colorectal Carcinoma, According to a Comprehensive Analysis.

A novel large neutral amino acid transporter 1 (LAT1)-specific inhibitor, JPH203, is expected to cause cancer-specific starvation and possess anti-tumor effects; however, its anti-tumor mechanism for colorectal cancer (CRC) remains unclear. We analyzed LAT family gene expressions in public databases using UCSC Xena and evaluated LAT1 protein expression using immunohistochemistry in 154 cases of surgically resected CRC. We also evaluated mRNA expression using polymerase chain reaction in 10 CRC cell lines. Furthermore, JPH203 treatment experiments were conducted in vitro and in vivo using an allogeneic immune-responsive mouse model with abundant stroma created via the orthotopic transplantation of the mouse-derived CRC cell line CT26 and mesenchymal stem cells. The treatment experiments were followed by comprehensive gene expression analyses with RNA sequencing. Database analyses and immunohistochemistry research on clinical specimens revealed that LAT1 expression was cancer-dominant, and its increase was accompanied by tumor progression. In vitro, JPH203 was effective in an LAT1 expression-dependent manner. In vivo, JPH203 treatment considerably reduced tumor size and metastasis, and RNA sequencing-based pathway analysis showed that not only tumor growth and amino acid metabolism pathways, but also stromal activation-related pathways were suppressed. The results of the RNA sequencing were validated in the clinical specimens, as well as both in vitro and in vivo. LAT1 expression in CRC plays an important role in tumor progression. JPH203 may inhibit the progression of CRC and tumor stromal activity.

Comprehensive Analysis of Gene Expression Profiling to Explore Predictive Markers for Eradication Therapy Efficacy against Helicobacter pylori-Negative Gastric MALT Lymphoma.

Although radiotherapy is the standard treatment for Helicobacter pylori (Hp)-negative gastric mucosa-associated lymphoid tissue (MALT) lymphoma, eradication therapy using antibiotics and an acid secretion suppressor can sometimes induce complete remission. We explored predictive markers for the response to eradication therapy for gastric MALT lymphoma that were negative for both API2-MALT1 and Hp infection using comprehensive RNA sequence analysis. Among 164 gastric MALT lymphoma patients who underwent eradication therapy as primary treatment, 36 were negative for both the API2-MALT1 fusion gene and Hp infection. Based on eradication therapy efficacy, two groups were established: complete response (CR) and no change (NC). The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that cancer-related genes and infection-related genes were highly expressed in the NC and CR groups, respectively. Based on this finding and transcription factor, gene ontology enrichment, and protein-protein interaction analyses, we selected 16 candidate genes for predicting eradication therapy efficacy. Real-time PCR validation in 36 Hp-negative patients showed significantly higher expression of olfactomedin-4 (OLFM4) and the Nanog homeobox (NANOG) in the CR and NC groups, respectively. OLFM4 and NANOG could be positive and negative predictive markers, respectively, for eradication therapy efficacy against gastric MALT lymphoma that is negative for both API2-MALT1 and Hp infection.

Anti-Programmed Cell Death-1 Antibody and Dasatinib Combination Therapy Exhibits Efficacy in Metastatic Colorectal Cancer Mouse Models.

In this study, we investigated the in vivo metastasis suppression effects of the platelet-derived growth factor receptor inhibitor dasatinib, which targets cancer-associated fibroblasts (CAFs), in combination with an anti-programmed cell death-1 (PD-1) antibody. We classified clinical CRC cases as inflamed, excluded, or desert using immunohistochemical analysis and evaluated the tumor stroma. The excluded type was the most common, and cases with high-volume stroma in the primary lesions also had a high stromal volume in the liver metastatic lesions. Liver-metastasis mouse models with different stromal volumes were established and treatment-induced changes in the tumor immune microenvironment were evaluated. The anti-PD-1 antibody alone exhibited a therapeutic effect for the liver metastases with low stromal volumes but not for the liver metastases with high stromal volumes. In contrast, antitumor effects were observed with anti-PD-1 antibody/dasatinib combination therapy even in the liver metastases with high stromal volumes. Combination therapy reduced the stromal volume, promoted immune cell infiltration, induced antitumor cytotoxic T-cell responses, activated antitumor immunity, and promoted tumor regression. These results suggest that CAFs play an important role in the immune evasion of CRC and that anti-PD-1 antibody/dasatinib combination therapy has potential as a treatment option for patients with metastatic CRC for whom immunotherapy alone is ineffective.

RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4.

Chromosomal translocation generates the MLL-AF4 fusion gene, which causes acute leukemia of multiple lineages. MLL-AF4 is a strong oncogenic driver that induces leukemia without additional mutations and is the most common cause of pediatric leukemia. However, establishment of a murine disease model via retroviral transduction has been difficult owning to a lack of understanding of its regulatory mechanisms. Here, we show that MLL-AF4 protein is post-transcriptionally regulated by RNA-binding proteins, including those of KHDRBS and IGF2BP families. MLL-AF4 translation is inhibited by ribosomal stalling, which occurs at regulatory sites containing AU-rich sequences recognized by KHDRBSs. Synonymous mutations disrupting the association of KHDRBSs result in proper translation of MLL-AF4 and leukemic transformation. Consequently, the synonymous MLL-AF4 mutant induces leukemia in vivo. Our results reveal that post-transcriptional regulation critically controls the oncogenic activity of MLL-AF4; these findings might be valuable in developing novel therapies via modulation of the activity of RNA-binding proteins.

A feline case of multiple myeloma treated with bortezomib.

BACKGROUND: Multiple myeloma (MM) is an uncommon neoplasm in cats. There is no established standard of treatment due to the rare occurrence of this disease in cats. Bortezomib is a proteasome inhibitor that serves as the first-line drug for MM in humans, but its effectiveness currently is unknown in feline MM. We present here the case report of a feline MM that exhibited a favorable response to bortezomib. CASE PRESENTATION: The case was an 11-year-old non-castrated male domestic cat with light-chain MM presenting with clinical symptoms (anorexia, fatigue, and vomiting), mild azotemia, and pancytopenia. The cat failed on melphalan with prednisolone (MP), so bortezomib (Velcade) was initiated on Day 88. A total of 6 cycles of the treatment was performed, with each treatment cycle consisting of twice-weekly subcutaneous administration for 2 weeks followed by a 1-week rest. The dose of bortezomib was 0.7 mg/m2 for first week and 1.0 mg/m2 for second week in the first cycle. A dose of 0.7 mg/m2 was used for subsequent cycles. Prednisolone was used concomitantly in the first 2 cycles. Following treatment with bortezomib, clinical symptoms disappeared and a decrease in serum globulin and recovery of pancytopenia were noted. A monoclonal gammopathy, overproduction of serum immunoglobulin light chain, and Bence-Jones proteinuria that existed at diagnosis were undetectable on Day 123. A monoclonal gammopathy also was not detectable at the end of the bortezomib treatment (Day 213). Anorexia, fatigue, and marked bone marrow toxicity were experienced when bortezomib was administrated at a dose of 1.0 mg/m2, while no recognizable toxicity was observed at a dose of 0.7 mg/m2 throughout the treatment period. The case was placed on follow-up and there was no evidence of relapse as of Day 243. CONCLUSIONS: Bortezomib was effective and durable for the treatment of this case of feline MM after failure with MP. Bortezomib was well-tolerated in this cat at a dose of 0.7 mg/m2, but not at 1.0 mg/m2. Bortezomib appears to be a drug worthy of further study for the treatment of feline MM.

Investigation of endoscopic findings in nine cases of Helicobacter suis-infected gastritis complicated by gastric mucosa-associated lymphoid tissue lymphoma.

BACKGROUND: We have previously reported that eradication therapy was more effective against Helicobacter pylori (Hp)-negative gastric mucosa-associated lymphoid tissue (MALT) lymphoma in non-Helicobacter pylori Helicobacter (NHPH)-positive cases than in NHPH-negative cases and that the infection status of NHPH could be a predictive marker for the efficacy of eradication therapy for H. pylori negative gastric MALT lymphoma. However, a diagnostic test for NHPH infection has not yet been clinically established. In this study, we investigated the endoscopic findings in cases of H. suis-infected gastritis associated with gastric MALT lymphoma reported at our institution. MATERIALS AND METHODS: Participants were selected from cases of gastric MALT lymphoma who underwent esophagogastroduodenoscopy at Hiroshima University Hospital, who were negative for the API2-MALT1 gene, and who received eradication therapy as a first-line treatment. We examined the endoscopic findings in nine cases from this group in which H. suis infection was confirmed by polymerase chain reaction. RESULTS: Endoscopic findings, such as cracked mucosa, spotty redness, nodular gastritis-like appearance, and white marbled appearance, which have been reported as characteristics of NHPH gastritis, were observed in multiple cases. The most common endoscopic findings in this study were cracked mucosa (7/9 cases), followed by spotty redness (6/9 cases), nodular gastritis-like appearance (5/9 cases), and white marbled appearance (2/9 cases). CONCLUSIONS: Our study may serve as a reference for re-evaluation of the diagnostic criteria for H. suis infection and indications for eradication therapy, particularly for cases of H. pylori negative gastric MALT lymphoma, where endoscopic findings such as those seen in this study were observed in the background mucosa.

Molecular characterization of canine SHP2 mutants and anti-tumour effect of SHP2 inhibitor, SHP099, in a xenograft mouse model of canine histiocytic sarcoma.

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic neoplasm. Mutations in src homology 2 domain-containing phosphatase 2 (SHP2; encoded by PTPN11), which recently have been identified in canine HS tumour cells, could be attractive therapeutic targets for SHP099, an allosteric inhibitor of SHP2. Here, molecular characteristics of wild-type SHP2 and four SHP2 mutants (p.Ala72Gly, p.Glu76Gln, p.Glu76Ala and p.Gly503Val), including one that was newly identified in the present study, were investigated. Furthermore, in vivo effects of SHP099 on a HS cell line carrying SHP2 p.Glu76Ala were examined using a xenograft mouse model. While SHP2 Glu76 mutant cell lines and SHP2 wild-type/Gly503 mutant cell lines are highly susceptible and non-susceptible to SHP099, respectively, a cell line carrying the newly identified SHP2 p.Ala72Gly mutation exhibited moderate susceptibility to SHP099. Among recombinant wild-type protein and four mutant SHP2 proteins, three mutants (SHP2 p.Ala72Gly, p.Glu76Gln, p.Glu76Ala) were constitutively activated, while no activity was detected in wild-type SHP2 and SHP2 p.Gly503Val. Activities of these constitutively activated proteins were suppressed by SHP099; in particular, Glu76 mutants were highly sensitive. In the xenograft mouse model, SHP099 showed anti-tumour activity against a SHP2 p.Glu76Ala mutant cell line. Thus, there was heterogeneity in molecular characteristics among SHP2 mutants. SHP2 p.Glu76Ala and perhaps p.Glu76Gln, but not wild-type SHP2 or SHP2 p.Gly503Val, were considered to be oncogenic drivers targetable with SHP099 in canine HS. Further studies will be needed to elucidate the potential of SHP2 p.Ala72Gly as a therapeutic target of SHP099 in canine HS.

HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis.

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.

HOXA9 promotes MYC-mediated leukemogenesis by maintaining gene expression for multiple anti-apoptotic pathways.

HOXA9 is often highly expressed in leukemias. However, its precise roles in leukemogenesis remain elusive. Here, we show that HOXA9 maintains gene expression for multiple anti-apoptotic pathways to promote leukemogenesis. In MLL fusion-mediated leukemia, MLL fusion directly activates the expression of MYC and HOXA9. Combined expression of MYC and HOXA9 induced leukemia, whereas single gene transduction of either did not, indicating a synergy between MYC and HOXA9. HOXA9 sustained expression of the genes implicated in the hematopoietic precursor identity when expressed in hematopoietic precursors, but did not reactivate it once silenced. Among the HOXA9 target genes, BCL2 and SOX4 synergistically induced leukemia with MYC. Not only BCL2, but also SOX4 suppressed apoptosis, indicating that multiple anti-apoptotic pathways underlie cooperative leukemogenesis by HOXA9 and MYC. These results demonstrate that HOXA9 is a crucial transcriptional maintenance factor that promotes MYC-mediated leukemogenesis, potentially explaining why HOXA9 is highly expressed in many leukemias.

Oncogenic mutations Q61L and Q61H confer active form-like structural features to the inactive state (state 1) conformation of H-Ras protein.

GTP-bound forms of Ras proteins (Ras•GTP) assume two interconverting conformations, "inactive" state 1 and "active" state 2. Our previous study on the crystal structure of the state 1 conformation of H-Ras in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp) indicated that state 1 is stabilized by intramolecular hydrogen-bonding interactions formed by Gln61. Since Ras are constitutively activated by substitution mutations of Gln61, here we determine crystal structures of the state 1 conformation of H-Ras•GppNHp carrying representative mutations Q61L and Q61H to observe the effect of the mutations. The results show that these mutations alter the mode of hydrogen-bonding interactions of the residue 61 with Switch II residues and induce conformational destabilization of the neighboring regions. In particular, Q61L mutation results in acquirement of state 2-like structural features. Moreover, the mutations are likely to impair an intramolecular structural communication between Switch I and Switch II. Molecular dynamics simulations starting from these structures support the above observations. These findings may give a new insight into the molecular mechanism underlying the aberrant activation of the Gln61 mutants.

Protocol for fractionation-assisted native ChIP (fanChIP) to capture protein-protein/DNA interactions on chromatin.

Conventional chromatin immunoprecipitation (ChIP) includes many steps that need to be optimized. Here, we have described a protocol of fractionation-assisted native ChIP (fanChIP) which combines subfractionation and native ChIP to purify protein/chromatin complexes applicable for analyses of both protein-protein and protein-DNA interactions within a short period of time. fanChIP is advantageous as subcellular fractionation removes chromatin-unbound materials before immunoprecipitation, and the chromatin fragmentation by micrococcal nuclease (MNase) in a mild condition enables one-step purification of intact protein/chromatin complexes. For complete details on the use and execution of this protocol, please refer to Miyamoto et al. (2020).

A canine case of malignant melanoma carrying a KIT c.1725_1733del mutation treated with toceranib: a case report and in vitro analysis.

BACKGROUND: Canine malignant melanoma is highly aggressive and generally chemoresistant. Toceranib is a kinase inhibitor drug that inhibits several tyrosine kinases including the proto-oncogene receptor tyrosine kinase KIT. Although canine malignant melanoma cells often express KIT, a therapeutic effect for toceranib has yet to be reported for this tumor, with only a small number of patients studied to date. This is a case report of a dog with malignant melanoma that experienced a transient response to toceranib. Furthermore, the KIT expressed in the tumor of this case was examined using molecular analysis. CASE PRESENTATION: A Shiba Inu dog presented with a gingival malignant melanoma extending into surrounding structures with metastasis to a submandibular lymph node. The dog was treated with toceranib (Palladia®; 2.6-2.9 mg/kg, orally, every other day) alone. Improvement of tumor-associated clinical signs (e.g., halitosis, tumor hemorrhage, trismus, and facial edema) with reduced size of the metastatic lymph node was observed on Day 15. The gingival tumor and associated masses in the masseter and pterygoid muscles decreased in size by Day 29 of treatment. Toceranib treatment was terminated on Day 43 due to disease progression and the dog died on Day 54. The tumor of this dog had a novel deletion mutation c.1725_1733del within KIT and the mutation caused ligand-independent phosphorylation of KIT, which was suppressed by toceranib. This mutation was considered to be an oncogenic driver mutation in the tumor of this dog, thereby explaining the anti-tumor activity of toceranib. CONCLUSIONS: This is the first report that presents a canine case of malignant melanoma that responded to toceranib therapy. KIT encoded by KIT harboring a mutation c.1725_1733del is a potential therapeutic target for toceranib in canine malignant melanoma. Further investigation of the KIT mutation status and toceranib therapy in canine malignant melanoma will need to be undertaken.

Commitment toward cell death by activation of autophagy with survivin inhibitor YM155 in two canine squamous cell carcinoma cell lines with high expression of survivin.

Canine squamous cell carcinoma (SCC) is difficult to treat if local therapy is not feasible. Recently, survivin inhibitor YM155 was shown to have growth inhibitory activity on high-survivin-expressing canine SCC cell lines HAPPY and SQ4. Here, the mechanisms underlying the effect of YM155 on these cell lines were investigated. YM155 induced cleavage of poly(ADP-ribose) polymerase (PARP) in HAPPY, but not in SQ4 cells. Analyzing two autophagy markers, the level of microtubule-associated protein 1 light chain 3 (LC3)-II and the LC3-II/LC3-I ratio, indicated that YM155 activates autophagy in both cell lines, and this activation occurs prior to PARP cleavage in HAPPY cells. Moreover, inhibition of autophagic flux by chloroquine almost completely prevented the toxic effect of YM155 in both cell lines. Although there are differences in their eventual cell death type, both cell lines may be committed to cell death by activation of autophagy with YM155. Activation of autophagy is likely to be a key mechanism in the growth-inhibitory effects of YM155 in these lines. These data provide new insights into the cytotoxic mechanism of YM155 in canine SCC cells.

Activation of CpG-Rich Promoters Mediated by MLL Drives MOZ-Rearranged Leukemia.

Uncontrolled self-renewal of hematopoietic progenitors induces leukemia. To self-renew, leukemia cells must continuously activate genes that were previously active in their mother cells. Here, we describe the circuitry of a transactivation system responsible for oncogenic self-renewal. MLL recruits RNA polymerase II (RNAP2) to unmethylated CpG-rich promoters by its CXXC domain and activates transcription by transcriptional regulators, including the AF4 family/ENL family/P-TEFb complex, DOT1L, and p300/CBP histone acetyl transferases. MOZ also targets a broad range of CpG-rich promoters through association with RNAP2 and MLL. Leukemic fusion proteins such as MOZ-TIF2 and MLL-AFX constitutively activate CpG-rich promoters by aberrantly recruiting p300/CBP. Pharmacological inhibition of MLL or DOT1L induces differentiation of MOZ-TIF2-transformed cells. These results reveal that activation of unmethylated CpG-rich promoters mediated by MLL is the central mechanism of oncogenic self-renewal in MOZ-rearranged leukemia and indicate that the molecularly targeted therapies intended for MLL-rearranged leukemia can be applied for MOZ-rearranged leukemia.

Foxp3+ Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs.

Regulatory T cells (Tregs) can be targeted in cancer immunotherapy. A previous study has shown that the chemokine CCL17 and the receptor CCR4 play a role in Treg recruitment in canine urothelial carcinoma. Here, we describe the association of tumor-infiltrating Tregs with CCL17/CCR4 expression in dogs with other carcinomas. In this study, we investigated 23 dogs with mammary carcinoma, 14 dogs with oral squamous cell carcinoma, 16 dogs with pulmonary adenocarcinoma, and 8 healthy control dogs. Immunohistochemistry showed that Foxp3+ Tregs and CCR4+ cells were increased in the tumor tissues of mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, when compared with the healthy tissues. The number of CCR4+ cells was associated with that of Foxp3+ Tregs. Double immunofluorescence labeling confirmed that most tumor-infiltrating Foxp3+ Tregs expressed CCR4. In vitro, canine carcinoma cell lines expressed CCL17 mRNA. Quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) showed that CCL17 mRNA expression in canine carcinomas was increased approximately 10- to 25-fold relative to that of healthy tissues. These results suggest that the CCL17/CCR4 axis may drive Treg recruitment in a variety of canine carcinomas. CCR4 blockade may be a potential therapeutic option for tumor eradication through Treg depletion.