FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

HSP90 Inhibitor PU-H71 in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the molecular chaperone HSP90 and the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The HSP90 inhibitor PU-H71, MCL1 inhibitor S63845, and BCL2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including FLT3-ITD and TP53 mutant AML cell lines and a variety of patient-derived AML cells. Results: PU-H71 and combination treatments with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in susceptible AML cell lines and primary AML. The majority of the primary AML samples were responsive to PU-H71 in combination with BH3 mimetics. Elevated susceptibility to PU-H71 and S63845 was associated with FLT3 mutated AML with CD34 < 20%. Elevated susceptibility to PU-H71 and venetoclax was associated with primary AML with CD117 > 80% and CD11b < 45%. The combination of HSP90 inhibitor PU-H71 and MCL1 inhibitor S63845 may be a candidate treatment for FLT3-mutated AML with moderate CD34 positivity while the combination of HSP90 inhibitor PU-H71 and BCL2 inhibitor venetoclax may be more effective in the treatment of primitive AML with high CD117 and low CD11b positivity.

[Correlation between expression of long non-coding RNA ZXF1 and prognosis of lung adenocarcinoma and its potential molecular mechanism].

Objective: To investigate the correlation between expression of long non-coding RNA (lncRNA) ZXF1 and clinicopathological characteristics, as well as prognosis of lung adenocarcinoma; and to explore its potential molecular mechanism. Methods: A total of 83 lung adenocarcinoma tissue samples and 83 paracancerous lung tissue samples from lung adenocarcinoma patients were collected. The mRNA expression of ZXF1 in the tumors and the corresponding adjacent non-tumor tissues were determined by real-time fluorescence quantitative PCR. The correlation between ZXF1 level and clinicopathological characteristics such as age, gender, smoking history, tumor size, tumor differentiation and lymph node metastasis was evaluated. Survival analysis was performed using Kaplan-Meier and Cox multivariate regression, based on the data of a 12-56 months follow-up after surgery. In vitro ZXF1 was over-expressed in lung adenocarcinoma A549 cells, and then the proteins functionally related to ZXF1 were identified by protein array analysis. Results: Of the 83 cases of lung adenocarcinoma, the ZXF1 mRNA levels in the tumor and adjacent non-tumor tissues were 8.32±3.05 and 1.05±0.47, respectively (P<0.05), and a high-level the high expression of ZXF1 in the tumor tissues was detected in 56 cases. The expression status of ZXF1 was closely correlated with the tumor differentiation and lymph node metastasis (P<0.05), but was not significantly related to age, gender and tumor size. Based on a 12-56 months follow-up, the patients with high level ZXF1 expression had a shorter disease free survival (DFS) and overall survival (OS) than that of the group with low level ZXF1 (all P<0.05). Multivariate analysis showed that ZXF1 expression, tumor size and lymph node metastasis were independent risk factors to DFS; and ZXF1 expression, tumor size, lymph node metastasis and tumor differentiation were independent risk factors to OS. The protein array data revealed that expressions of bone morphogenetic protein 5 (BMP-5)and stem cell factor receptor (SCFR)were upregulated upon overexpression of the ZXF1 in A549 cells. Conclusions: lncRNA ZXF1 is overexpressed in lung adenocarcinoma, and is closely correlated with tumor differentiation and lymph node metastasis. As an independent risk factor, a high expression of ZXF1 indicates a poor prognosis for the patients. ZXF1 may influence the biological behavior of lung adenocarcinoma by enhancing the protein expression of BMP-5 and SCFR.

CRISPR/Cas9-engineered human ES cells harboring heterozygous and homozygous c-KIT knockout.

The receptor tyrosine kinase c-KIT (CD117) has a key role in hematopoiesis and is a marker for endothelial and cardiac progenitor cells. In vivo, deficiency of c-KIT is lethal and therefore using CRISPR/Cas9 editing we generated heterozygous and homozygous c-KIT knockout human embryonic stem cell (ES cell) lines. The c-KIT knockout left ES cell pluripotency unaffected as shown by immunofluorescence and trilineage differentiation potential. Heterozygous and homozygous c-KIT knockouts showed complete loss of exon 17, resulting in ablation of c-KIT protein from the cell surface. c-KIT knockout ES cells provide a valuable tool for further investigating c-KIT biology.

GIST: Correlation of risk classifications and outcome.

In clinical practice, there are often discrepancies between the oncological prognosis of gastrointestinal stromal tumors (GIST) and the actual clinical course. This study aimed to check with our collective how reliably the current classifications (Miettinen, Fletcher) predict the prognosis of GIST and to evaluate whether an extension of the classifications by the parameter proliferation activity could make sense. This prospective study enrolled 58 patients who underwent surgery on GIST from 01/2006 to 12/2016. The postoperative course (curation, recurrence, progress) was correlated with the identified risk classification and the proliferative activity. Coincidences with other tumors were strikingly common in patients with GIST (43%). Based on the risk group assignment of GIST, no assessment of the probability of the occurrence of second neoplasia could be derived. Individual patients were under- or over-graduated concerning the assessment of biological behavior based on the standard risk classifications. The inclusion of proliferative activity did not allow for a more precise predictive power - neither to the risk of recurrence and metastasis nor to the development of a second neoplasia. The study showed that there is currently no parameter or logarithm that reliably predicts the biological behavior of GIST. Due to the frequency of coincidence of second neoplasia and (rare) distant metastases, for everyday clinical practice, appropriate staging diagnostic and regular follow-up care should also be used for benign GIST.

C-kit is important for SOD1(G93A) mouse survival independent of mast cells.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease leading to progressive and lethal paralysis. The disease process is multi-factorial and is characterized by selective motor neuron degeneration. Previous work demonstrated that the local concentration of various growth factors can influence motor neuron survival and disease progression. A potential role for c-kit, a growth factor receptor present in the spinal cord, in ALS is unknown. To dissect the role of c-kit in ALS we interbred SOD1(G93A) mice with kit(w-sh/w-sh) mice, which have a 70% decrease in c-kit expression in the spinal cord. kit(w-sh/w-sh) SOD1(G93A) mice have a reduced survival compared to SOD1(G93A) mice, while the amount of motor neurons at end stage is similar. By means of grip strength and nerve conductance analysis we show that kit(w-sh/w-sh) mice have diminished strength and slightly impaired compound muscle action potential latency, although the number of neurons is similar across genotypes. Decreasing kit gene expression in SOD1(G93A) mice is detrimental and our results imply that this effect is independent of mast cells, as tested by ketotifen administration. To conclude, our data expand on the protective role of growth factors in ALS, as decreasing c-kit by approximately 70% is detrimental in SOD1(G93A) mice.

Erythropoietin acts as an anti-inflammatory signal on murine mast cells.

Recently it was found that the erythropoietin receptor (EpoR) is expressed on innate immune cells, such as dendritic cells and macrophages. We found that murine bone marrow-derived mast cells express the EpoR and that its expression is increased under hypoxic conditions. Interestingly, Epo stimulation of the cells did not activate signal transducer and activator of transcription molecules, nor did we find differences in the expression of typical STAT-dependent genes, the proliferation rate, and the ability to differentiate or to protect the cells from apoptosis. Instead, we demonstrate that stimulation of mast cells with Epo leads to phosphorylation of the receptor tyrosine kinase c-kit. We hypothesize that this is due to the formation of a receptor complex between the EpoR and c-kit. The common beta chain of the IL-3 receptor family, which was described as part of the tissue protective receptor (TPR) on other non-erythroid cells, however is not activated. To investigate whether the EpoR/c-kit complex has tissue protective properties, cells were treated with the Toll-like receptor ligand LPS. Combined Epo and LPS treatment downregulated the inflammatory response of the cells as detected by a decrease in IL-6 and TNF-α secretion.

Gene-expression profiling elucidates molecular signaling networks that can be therapeutically targeted in vestibular schwannoma.

OBJECT: Vestibular schwannomas (VS) are common benign tumors of the vestibular nerve that cause significant morbidity. The current treatment strategies for VS include surgery or radiation, with each treatment option having associated complications and side effects. The transcriptional landscape of schwannoma remains largely unknown. METHODS: In this study the authors performed gene-expression profiling of 49 schwannomas and 7 normal control vestibular nerves to identify tumor-specific gene-expression patterns. They also interrogated whether schwannomas comprise several molecular subtypes using several transcription-based clustering strategies. The authors also performed in vitro experiments testing therapeutic inhibitors of over-activated pathways in a schwannoma cell line, namely the PI3K/AKT/mTOR pathway. RESULTS: The authors identified over 4000 differentially expressed genes between controls and schwannomas with network analysis, uncovering proliferation and anti-apoptotic pathways previously not implicated in VS. Furthermore, using several distinct clustering technologies, they could not reproducibly identify distinct VS subtypes or significant differences between sporadic and germline NF2-associated schwannomas, suggesting that they are highly similar entities. The authors identified overexpression of PI3K/AKT/mTOR signaling networks in their gene-expression study and evaluated this pathway for therapeutic targeting. Testing the compounds BEZ235 and PKI-587, both novel dual inhibitors of PI3K and mTOR, attenuated tumor growth in a preclinical cell line model of schwannoma (HEI-293). In vitro findings demonstrated that pharmacological inhibition of the PI3K/AKT/mTOR pathway with next-generation compounds led to decreased cell viability and increased cell death. CONCLUSIONS: These findings implicate aberrant activation of the PI3K/AKT/mTOR pathway as a molecular mechanism of pathogenesis in VS and suggest inhibition of this pathway as a potential treatment strategy.

Combination antiangiogenic therapy and radiation in head and neck cancers.

Tumor angiogenesis is a hallmark of advanced cancers and promotes invasion and metastasis. Over 90% of head and neck squamous cell carcinomas (HNSCC) express angiogenic factors such as vascular endothelial growth factor (VEGF). Several preclinical studies support the prognostic implications of angiogenic markers for HNSCC and currently this is an attractive treatment target in solid tumors. Since radiotherapy is one of the most commonly used treatments for HNSCC, it is imperative to identify the interactions between antiangiogenic therapy and radiotherapy, and to develop combination therapy to improve clinical outcome. The mechanisms between antiangiogenic agents and ionizing radiation are complicated and involve many interactions between the vasculature, tumor stroma and tumor cells. The proliferation and metastasis of tumor cells rely on angiogenesis/blood vessel formation. Rapid growing tumors will cause hypoxia, which up-regulates tumor cell survival factors, such as hypoxia-inducing factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), giving rise to more tumor proliferation, angiogenesis and increased radioresistance. Thus, agents that target tumor vasculature and new tumor vessel formation can modulate the tumor microenvironment to improve tumor blood flow and oxygenation, leading to enhanced radiosensitivity. In this review, we discuss the mechanisms of how antiangiogenic therapies improve tumor response to radiation and data that support this combination strategy as a promising method for the treatment of HNSCC in the future.

Is exon mutation analysis needed for adjuvant treatment of gastrointestinal stromal tumor?

Gastrointestinal stromal tumors (GISTs) are the most common soft tissue sarcoma of the gastrointestinal tract, resulting from an activating mutation of stem cell factor receptor (KIT), and an activating mutation of the homologous platelet-derived growth factor receptor alpha (PDGFRA) kinase. Most GISTs (90%-95%) are KIT-positive. About 5% of GISTs are truly negative for KIT expression. GISTs have been documented to resistant conventional chemotherapeutics. Due to the KIT activation that occurs in the majority of the cases, KIT inhibition is the primary treatment approach in the adjuvant treatment of metastatic GISTs. Imatinib mesylate is an oral agent that is a selective protein tyrosine kinase inhibitor of the KIT protein tyrosine kinase, and it has demonstrated clinical benefit and objective tumor responses in most GIST patients in phase II and III trials. The presence and the type of KIT or PDGFRA mutation are predictive of response to imatinib therapy in patients with advanced and metastatic disease. Molecular analysis in phase I-II trials revealed significant differences in objective response, progression-free survival, and overall survival between GISTs with different kinase mutations. The aim of this letter is to touch on the need for exon mutation analysis for adjuvant treatment with imatinib in GIST patients.

Randomized phase II study of sunitinib versus standard of care for patients with previously treated advanced triple-negative breast cancer.

PURPOSE: This randomized, open-label phase II study compared the efficacy of sunitinib monotherapy with that of single-agent standard-of-care (SOC) chemotherapy in patients with previously treated advanced triple-negative breast cancer (TNBC). METHODS: Patients with advanced TNBC, relapsed after anthracycline- and taxane-based chemotherapy, were randomized to receive either sunitinib (37.5 mg/day) or the investigator's choice of SOC therapy. Progression-free survival was the primary endpoint. RESULTS: Median progression-free survival was 2.0 months with sunitinib and 2.7 months with SOC chemotherapy (one-sided P = 0.888). Median overall survival was not prolonged with sunitinib (9.4 months) compared with SOC chemotherapy (10.5 months; one-sided P = 0.839). The objective response rate was 3% with sunitinib and 7% with SOC chemotherapy (one-sided P = 0.962). CONCLUSIONS: Sunitinib monotherapy did not improve efficacy compared with SOC chemotherapy in patients with previously treated advanced TNBC, for which identification of effective treatments and therapeutic targets remains an urgent need. TRIAL REGISTRATION: NCT00246571.