Effects of Sorafenib, a Tyrosin Kinase Inhibitor, on Adrenocortical Cancer

The lack of an effective medical treatment for adrenocortical carcinoma (ACC) has prompted the search for better treatment protocols for ACC neoplasms. Sorafenib, a tyrosine kinase inhibitor has exhibited effectiveness in the treatment of different human tumors. Therefore, the aim of this study was to understand the mechanism through which sorafenib acts on ACC, especially since treatment with sorafenib alone is sometimes unable to induce a long-lasting antiproliferative effect in this tumor type. The effects of sorafenib were tested on the ACC cell line H295R by evaluating cell viability, apoptosis and VEGF receptor signaling which was assessed by analyzing VE-cadherin and β-catenin complex formation. We also tested sorafenib on an in vitro 3D cell culture model using the same cell line. Apoptosis was observed after sorafenib treatment, and coimmunoprecipitation data suggested that the drug prevents formation VEGFR-VE-cadherin and β-catenin proteins complex. These results were confirmed both by ultrastructural analysis and by a 3D model where we observed a disaggregation of spheres into single cells, which is a crucial event that represents the first step of metastasis. Our findings suggest that although sorafenib induces apoptotic cell death a small portion of cells survive the treatment and have characteristics of a malignancy. Based on our data we recommend against the use of sorafenib in patients with ACC.

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The Novel Cyclin-Dependent Kinase Inhibitor Flavopiridol Downregulates Bcl-2 and Induces Growth Arrest and Apoptosis in Chronic B-Cell Leukemia Lines

Blood ◽

10.1182/blood.v90.11.4307 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4307-4312 ◽

Cited By ~ 126

Author(s):

Andrea König ◽

Gary K. Schwartz ◽

Ramzi M. Mohammad ◽

Ayad Al-Katib ◽

Janice L. Gabrilove

Keyword(s):

Cell Line ◽

Kinase Inhibitor ◽

Chromatin Condensation ◽

Human Tumor ◽

Trypan Blue Exclusion ◽

Clinical Phase ◽

Cellular Effects ◽

Barr Virus

Abstract Flavopiridol is a novel, potent inhibitor of cyclin-dependent kinases (CDK). This synthetic flavone has been reported to exhibit antitumor activity in murine and human tumor cell lines in vitro and in vivo and is currently undergoing clinical phase I evaluation. In the present study, 1 Epstein-Barr virus (EBV)-transformed B-prolymphocytic cell line (JVM-2), 1 EBV-transformed B-CLL cell line (I83CLL), and 1 non-EBV transformed B-CLL cell line (WSU-CLL) were used as targets. Treatment of the cells with flavopiridol (100 nmol/L to 400 nmol/L) led to a marked dose- and time-dependent inhibition of cell growth and survival as determined using trypan blue exclusion. Morphologic analysis showed characteristic apoptotic changes such as chromatin condensation and fragmentation, membrane blebbing, and formation of apoptotic bodies. Furthermore, quantitative assessment of apoptosis-associated DNA strand breaks by in situ TdT labeling showed that a significant number of flavopiridol-treated cells underwent apoptosis. These cellular effects were associated with a significant decrease in bcl-2 expression as observed by Northern and Western blotting. The results showed that flavopiridol downregulates bcl-2 mRNA and bcl-2 protein expression within 24 hours. Genistein and quercetin, two flavonoids that do not inhibit CDKs, did not affect bcl-2 expression. These data suggest an additional mechanism of action of this new flavone which might be useful as an agent in the treatment of chronic lymphoid malignancies.

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Molecular Imaging of Nonsmall Cell Lung Carcinomas Expressing Active Mutant EGFR Kinase Using PET with [124I]-Morpholino-IPQA

BioMed Research International ◽

10.1155/2013/549359 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 4

Author(s):

Skye Hsin-Hsien Yeh ◽

Chien-Feng Lin ◽

Fan-Lin Kong ◽

Hsin-Ell Wang ◽

Ya-Ju Hsieh ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Water Solubility ◽

Kinase Inhibitor ◽

Control Cell ◽

H1299 Cell ◽

Subcutaneous Tumor ◽

Tumor Xenografts ◽

H1299 Cell Line

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) have high levels of basal receptor phosphorylation and are associated with clinical responsiveness to Iressa in patients with nonsmall cell lung cancer (NSCLC). This study aimed to assess the feasibility of morpholino-[124I]IPQA derivative as anin vivoPET imaging tool for the expression of different EGFR mutants in NSCLC.In vitroradiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention after washout of morpholino-[131I]IPQA derivative in high EGFR-expressing H1299 NSCLC derivative cell lines (L858R and E746-A750 del cell lines), but not in EGFR-transfected H1299 cell line and vector-transfected H1299 cell line. Using the morpholino-[124I]IPQA derivative, we obtained noninvasive microPET images of EGFR activity in L858R and E746-A750 del subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown form control cell line. Different EGFR mutant (activity) tumors have a different morpholino-[∗I]IPQA derivative uptake. However, it still needs to modify the structure of IPQA to increase its water solubility and reduce hepatobiliary clearance. Morpholino-[124I]IPQA derivative may be a potential probe for selection of the candidate patients suffering from NSCLC for the small molecule tyrosine kinase inhibitor therapy (e.g., Iressa) in the future.

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Rho-kinase inhibitor, fasudil, suppresses glioblastoma cell line progression in vitro and in vivo

Cancer Biology & Therapy ◽

10.4161/cbt.9.11.11634 ◽

2010 ◽

Vol 9 (11) ◽

pp. 875-884 ◽

Cited By ~ 49

Author(s):

Lin Deng ◽

Gang Li ◽

Ronghui Li ◽

Qinglin Liu ◽

Qiaowei He ◽

...

Keyword(s):

Cell Line ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Glioblastoma Cell Line ◽

Glioblastoma Cell ◽

Rho Kinase Inhibitor

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Preclinical Rationale for the Use of the Combined Treatment with the AKT Inhibitor Perifosine and the Multikinase Inhibitor Sorafenib in Hodgkin Lymphoma

Blood ◽

10.1182/blood.v118.21.1653.1653 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1653-1653

Author(s):

Silvia Locatelli ◽

Arianna Giacomini ◽

Anna Guidetti ◽

Loredana Cleris ◽

Michele Magni ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Cell Line ◽

Cell Lines ◽

Drug Combination ◽

Akt Inhibitor ◽

Sorafenib Treatment ◽

Responsive Cell

Abstract Abstract 1653 Introduction: A significant proportion of Hodgkin lymphoma (HL) patients refractory to first-line chemotherapy or relapsing after autologous transplantation are not cured with currently available treatments and require new treatments. The PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL. These pathways can be targeted using the AKT inhibitor perifosine (Æterna Zentaris GmBH, Germany, EU), and the RAF/MEK/ERK inhibitor sorafenib (Nexavar®, Bayer, Germany, EU). We hypothesized that perifosine in combination with sorafenib might have a therapeutic activity in HL by overcoming the cytoprotective and anti-apoptotic effects of PI3K/Akt and RAF/MEK/ERK pathways. Since preclinical evidence supporting the anti-lymphoma effects of the perifosine/sorafenib combination are still lacking, the present study aimed at investigating in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of apoptosis. In responsive cell lines, WB analysis showed that anti-proliferative events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. CONCLUSIONS: Perifosine/sorafenib combination resulted in potent anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation in HL patients. Disclosures: No relevant conflicts of interest to declare.

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Partially or Fully BCR-ABL Independent Mechanisms of in Vitro Resistance to Ponatinib

Blood ◽

10.1182/blood.v118.21.2481.2481 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2481-2481 ◽

Cited By ~ 1

Author(s):

Qian Yu ◽

Anna M Eiring ◽

Matthew S. Zabriskie ◽

Jamshid Khorashad ◽

David J Anderson ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Kinase Inhibitor ◽

Synthetic Lethality ◽

Immunoblot Analysis ◽

Kinase Domain ◽

Resistant Cell ◽

Abl Kinase ◽

Kinase Domain Mutation

Abstract Abstract 2481 Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs. Methods: Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib. Results: The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib (<5 nM) in the 32D(BCR-ABL)R cell line, yet these cells remained viable in the presence of up to 24 nM ponatinib. The effects of providing a second kinase inhibitor along with ponatinib (24 nM) in order to probe for synthetic lethality are under study. Possible involvement of a second, moderately ponatinib-sensitive target is suggested by the sharp ponatinib maximum at 24 nM; even 26 nM ponatinib is toxic to 32D(BCR-ABL)R cells. Thus far, ENU mutagenesis screens in human CML cell lines failed to yield resistant clones and only a few were recovered from the murine 32D(BCR-ABL)R cell line (3/1440 wells; the only BCR-ABL mutant recovered was BCR-ABLL387F). Conclusions: The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells. Disclosures: No relevant conflicts of interest to declare.

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In Vitro Anti-Myeloma Activity of the Multitargeted Kinase Inhibitor Midostaurin in the Context of Heterotypic Cocultures of Myeloma Cells with Nonmalignant Microenvironmental Accessory Cells

Blood ◽

10.1182/blood.v118.21.2923.2923 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2923-2923

Author(s):

Panisinee Lawasut ◽

Hannah M. Jacobs ◽

Jake E Delmore ◽

Joseph Negri ◽

Douglas W. McMillin ◽

...

Keyword(s):

Drug Resistance ◽

Research Funding ◽

Kinase Inhibitor ◽

Conditioned Media ◽

Myelogenous Leukemia ◽

Tumor Type ◽

Accessory Cells ◽

The Impact ◽

Positive Controls

Abstract Abstract 2923 Midostaurin (PKC412; Novartis Pharmaceuticals) is a multi-targeted kinase inhibitor currently being evaluated in clinical trials in acute myelogenous leukemia (AML), because of its potent activity in cells expressing mutant FLT3. Prior preclinical studies from our groups have shown that PKC412 has FLT3-independent anti-MM activity, and the effects on AML cells is suppressed by the presence of conditioned media from bone marrow stromal cells (BMSCs), such as the immortalized BMSC line HS-5 (Weisberg et al. Mol Cancer Ther 2007). In this study, we evaluated whether the microenvironment-dependent drug resistance to PKC412 applies to not only AML cells, but also to cells from MM and other FLT3-negative malignant cells. We tested a panel of cells from MM (n=8), FLT-ITDneg AML (n=1), CML (n=2) and breast cancer (n=2) for their response to PKC412 in the presence or absence of BMSCs and other non-malignant accessory cells using tumor cell compartment-specific bioluminescence imaging (CS-BLI), as in our antecedent studies (McMillin et al. Nat Medicine 2010). We also compared the PKC412 response of the aforementioned neoplastic cells when cultured in vitro in the presence or absence of conditioned media (CM) from different types of BMSCs known to confer PKC412 resistance in FLT3-mutant AML cells. Consistent with our previous studies of PKC412 treatment in conventional cultures of MM cells in isolation, we observed that PKC412 exhibits an anti-proliferative effect within the first 24 hrs of treatment, with major reduction of the numbers of viable cells at 48 and 72 hrs. At sub-micromolar doses that did not significantly affect the viability of non-malignant accessory cells tested, PKC412 had similar (or for some MM cell lines had more pronounced) activity against the MM cells, both in the presence and absence of the non-malignant accessory cells tested (HS-5, HS-27a, NIH-3T3 cells with or without transfection with human CD40L, etc.). In contrast, under the same experimental conditions, coculture with either BMSCs or exposure to their conditional media, decreased the response of MM cells to dexamethasone. These results suggested in contrast to the impact on FLT3mut AML, that the anti-MM activity of PKC412 is preserved (and in some cases slightly enhanced) when the MM cells interact with microenvironmental accessory cells and/or their secreted growth/survival factors. To obtain insight on possible mechanistic foundations of these observations, we examined the pattern of kinases inhibited by PKC412 at sub-μM concentrations (using FLT3 and FGFR3, known targets of PKC412 as positive controls). The results of in vitro kinase activity assays showed that PKC412 potently suppresses the aforementioned positive controls, but also exerts >50% inhibitory effect on the in vitro activity of additional kinases such as Akt2, Pim1, GSK3a, PDK1, p70S6K, SRC and Aurora A. Many of these kinases are known to participate in proliferative/anti-apoptotic signaling cascades downstream of cytokine/growth factor receptors or cell adhesion-mediated events triggered during MM–stromal interactions. We therefore conclude that the influence of the tumor microenvironment on the anti-neoplastic effects of PKC412 may be tumor-type dependent. The anti-MM activity of PKC412 is not subject to drug resistance triggered by non-malignant accessory cells, and conversely is occasionally moderately enhanced by these MM-stromal interactions. Mechanistically, this observation may be attributed in part to the multi-targeted nature of this inhibitor and, in particular, its aggregate impact on several kinases known to mediate stroma-induced proliferative and antipoptotic signaling in MM. Disclosures: Griffin: Novartis Pharmaceuticals: Consultancy, Research Funding. Richardson:Millennium: ; Celgene: ; Johnson & Johnson: ; Novartis: ; Bristol Myers Squibb:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Mitsiades:EMD Serono: Research Funding; AVEO Pharma: Research Funding; Amgen: Research Funding; OSI Pharmaceuticals: Research Funding; PharmaMar: licensing royalties; Amnis Therapeutics: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.

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Single Cell RNA-Seq Characterises Pre-Leukemic Transformation Driven By CEBPA N321D in the Hoxb8-FL Cell Line

Blood ◽

10.1182/blood-2018-99-110626 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3887-3887

Author(s):

Moosa Qureshi ◽

Fernando Calero-Nieto ◽

Iwo Kucinski ◽

Sarah Kinston ◽

George Giotopoulos ◽

...

Keyword(s):

Dendritic Cell ◽

Single Cell ◽

Cell Line ◽

Single Cells ◽

Mutant Form ◽

Rna Seq ◽

Wild Type ◽

Leukemic Transformation

Abstract The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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Optogenetic activators of apoptosis, necroptosis and pyroptosis for probing cell death dynamics and bystander cell responses

10.1101/2021.08.31.458313 ◽

2021 ◽

Author(s):

Kateryna Shkarina ◽

Eva Hasel de Carvalho ◽

José Carlos Santos ◽

Maria Leptin ◽

Petr Broz

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Single Cells ◽

Proof Of Concept ◽

Cell Responses ◽

Or Groups ◽

Wide Range ◽

Specific Induction

AbstractTargeted and specific induction of cell death in individual or groups of cells holds the potential for new insights into the response of tissues or organisms to different forms of death. Here we report the development of optogenetically-controlled cell death effectors (optoCDEs), a novel class of optogenetic tools that enables light-mediated induction of three types of programmed cell death (PCD) – apoptosis, pyroptosis and necroptosis – using Arabidopsis thaliana photosensitive protein Cryptochrome2. OptoCDEs enable rapid and highly specific induction of PCD in human, mouse and zebrafish cells and are suitable for a wide range of applications, such as sub-lethal cell death induction or precise elimination of single cells or cell populations in vitro and in vivo. As the proof-of-concept, we utilize optoCDEs to assess the differences in the neighboring cell response to apoptotic or necrotic PCD, revealing a new role for shingosine-1-phosphate signaling in regulating the efferocytosis of apoptotic cell by epithelia.

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Dual Targeting of JAK2 Signaling with a Type II JAK2 Inhibitor and of mTOR with a TOR Kinase Inhibitor Induces Apoptosis in CRLF2-Rearranged Ph-like Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.3706.3706 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3706-3706 ◽

Cited By ~ 1

Author(s):

Ce Shi ◽

Lina Han ◽

Yoko Tabe ◽

Hong Mu ◽

Shuo-Chieh Wu ◽

...

Keyword(s):

Cell Death ◽

Intracellular Signaling ◽

Kinase Inhibitor ◽

Apoptotic Cell ◽

Lymphoblastic Leukemia ◽

Mtor Inhibitors ◽

Type I ◽

Type Ii ◽

Jak2 Inhibitor

Abstract Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a subtype of high-risk B-precursor ALL (B-ALL) that carries a high risk of relapse after conventional chemotherapy (Mullighan et al, N Engl J Med. 2009). Rearrangements in CRLF2, leading to overexpression of the receptor for the cytokine thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALLs and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010; Tasian et al, Blood 2014). Previous studies established that combining a tyrosine kinase inhibitor (TKI) with an mTOR inhibitor provides greater anti-leukemia efficacy than a TKI alone in Ph+ B-ALL (Janes et al, Nat. Med. 2013). While allosteric mTOR inhibitors such as rapamycin only partially block mTORC1 and do not directly inhibit mTORC2, second-generation ATP-competitive mTOR kinase inhibitors (TOR-KIs) efficiently block both mTOR outputs and show greater efficacy when combined with TKIs. In this study, we investigated anti-leukemia efficacy and intracellular signaling networks in Ph-like CRLF2+ ALL models treated with combinations of a type I or type II JAK-2 inhibitor and a TOR-KI. The inhibitors were tested in human B-precursor Ph-like ALL cell lines MUTZ5 (IGH@-CRLF2 translocation, JAK2 R683G mutation) and MHH-CALL-4 (IGH@-CRLF2 translocation, JAK2 I682F mutation), B-ALL cell line REH (CRLF2wt), and primary CRLF2+ xenograft cells in vitro. For signaling and growth inhibition studies, cells were stimulated with 25 ng IL-7 or TSLP for 30 min, then with JAK2 type I inhibitor ruxolitinib (500nM) or type II inhibitor NVP-BBT594 (500nM) (Andraos et al., Cancer Discov. 2012) and allosteric mTOR inhibitor rapamycin or TOR-KI AZD2014. Effects on intracellular signaling were determined by phospho-flow cytometry. Anti-leukemia effects were characterized by viable cell counts and annexin V flow cytometry. In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling (p-JAK2(Tyr1008), p-STAT5(Ty694)) and AKT/pS6 signaling (p-AKT(Ser473), p-rS6(S235/236) (Fig. 1A). Stimulation with IL-7, mimicking support by the normal bone marrow environment, induced a lesser degree of activation of these phospho-proteins, except for p-4EBP1(T37/46), which was constitutively highly expressed in these cells and further induced by IL-7. These findings warranted combination studies of JAK2 and mTOR inhibitors. JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, and S6 activation, yet failed to decrease p-4EBP1 (Fig. 1A). AZD2014 but not rapamycin fully inhibited p-4EBP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth arrest in CRLF2+ cells (Fig. 1A, C). In turn, combination of ruxolitinib and AZD2014 further reduced cell proliferation but did not induce apoptotic cell death (Fig. 1B, D). Recent studies indicate persistence of JAK2-mutated cells in myeloproliferative neoplasms upon long-term exposure to a type I JAK2 inhibitor, mediated by JAK2 heterodimerization and reactivation of JAK-STAT signaling (Koppikar et al., Nature 2012). We therefore compared the in vitro efficacy of ruxolitinib and BBT594, a type II JAK2 inhibitor that retains the ability to bind inactive JAK2, in Ph-like ALL cells. In MUTZ-5 but not in MHH-CALL-4 cells, ruxolitinib increased JAK2 activation loop phosphorylation (p-JAK2-Tyr1008) despite suppression of STAT5 phosphorylation; in contrast, BBT594 diminished both p-JAK2 and p-STAT5. Unexpectedly, BBT594 induced apoptotic cell death in both MUTZ5, MHH-CALL-4 (Fig 1B) and in ALL blasts recovered from primary CRLF2+ xenograft and grown in OP9 in vitro co-culture; the combination of BBT594 with AZD2014 increased apoptosis and reduced cell viability even further, in both cell lines and in stroma-attached primary ALL cells. In summary, these results suggest that efficient blockade of JAK2/STAT5 with a type II JAK2 inhibitor translates into cell death of JAK2-addicted CRLF2-rearranged cells and may have the capacity to eliminate JAK2-mutated clones. Concomitant blockade of TORC1 signaling with a TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4EBP1 and causes synthetic lethality, providing avenues for novel, rationally designed combinatorial regimens in this subset of Ph-like B-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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