Search

The research study Depicts the recent developments and demonstrates the growing power of online Bodies or we can say Communities in building brand reputation and customer relationships. The study also examines the role and impact of the social media sites on generating potential online traffic Online Marketing is becoming a promising industry with rapid career growth, increasing influences, and widespread activities As we know that the access to the internet over a larger population is on a rises, Soon the India is to witness a revolution India will soon rank within the top markets in the world with the greatest number of internet users due to the nation's fast digitization. Online marketing is becoming a distinct business niche. The technicalities of the internet are growing as more marketers choose to build a brand online and connect with audiences across the world. sites, blogs, and advertising Campaigns, SEO tactics, and other techniques have delved deeply into the technological metrics affecting one's online visibility. In India, online or digital advertising is growing at a rate of 25–30% annually. India also has the World’s largest Facebook population. The use of digital marketing has widened as a result of quick digitization, through websites, networking methods, etc. Internet usage is commonplace. It has significantly influenced the strength and magnitude of the growth of digital channels through rising the use of laptops, computers, and mobile devices. Whether utilized for social networking, informative Google searches, or making purchases, India has transformed this medium into a totally-fledged business. And as a result of the market's growth, Digital Marketing has emerged as a key method of attracting customers online. The market for gadgets like smartphones, computers, tablets, and other devices is expanding, which has only accelerated usage and expanded the market's target demographic. Internet material has a significant impact on people's lives. Display of thoughts through words, photographs, films, and other media has a solid foundation in people's thoughts, which transfers into their daily lives. In order to boost a brand's influence on the audience, digital marketing focuses heavily on using content to influence the audience online. The amount of time people spend online enables web marketers to develop effective tactics to change customer behavior. The appropriate approach turns the audience into customers. Online marketing is becoming a distinct business niche. The complexities around the web are growing as more marketers choose to build a brand image online and engage with audiences across the world. Websites, blogs, marketing initiatives, SEO tactics, etc. have deeply impacted the technical metrics affecting one's visibility on the internet.

Supplementary Table S1. AML patients. Supplementary Table S2. RT-qPCR primers. Supplementary Table S3. IC50 concentrations (nM) of FLT3 inhibitors, PP2A activators and GSK-3beta inhibitors. Supplementary Figure S1. Concurrent PP2A-activating drug treatment increases cytotoxicity of FLT3 inhibitors in Ba/F3-ITD cells with FLT3-ITD and in an MV4-11 orthotopic mouse model. Supplementary Figure S2. Chou-Talalay analysis of FLT3-WT cell lines. Supplementary Figure S3. Images of all mice in the in vivo experiment. Supplementary Figure S4. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor does not induce apoptosis in wild-type FLT3 AML, AML complete remission (CR) or AML complete remission with incomplete platelet recovery (CRp) marrows. Supplementary Figure S5. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor downregulates c-Myc and Pim-1 protein expression in cells with FLT3-ITD. Supplementary Figure S6. p-c-Myc (T58) and p-c-Myc (S62) expression in Ba/F3-ITD and MV4-11 cells treated with gilteritinib and/or FTY720. Supplementary Figure S7. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor produces variable changes in c-Myc and Pim-1 mRNA expression in cells with FLT3-ITD. Supplementary Figure S8. c-Myc and Pim-1 expression in FLT3-WT AML blasts treated with gilteritinib and/or FTY720. Supplementary Figure S9. Proteasome inhibition inhibits downregulation of c-Myc and Pim-1 expression by concurrent PP2A-activating drug and FLT3 inhibitor treatment. Supplementary Figure S10. Concurrent PP2A-activating drug and FLT3 inhibitor treatment increases c-Myc and Pim-1 protein turnover in cells with FLT3-ITD. Supplementary Figure S11. Concurrent PP2A activator and FLT3 inhibitor treatment downregulates p-ERK later than p-AKT in cells with FLT3-ITD. Supplementary Figure S12. The GSK-3beta inhibitor TWS119 prevents c-Myc and Pim-1 downregulation and apoptosis induction by PP2A-activating drug and FLT3 inhibitor combination in Ba/F3-ITD cells.

Fms-like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD–expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD–expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo. PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3β, and GSK-3β inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3β activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3β also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD–expressing cells through a novel mechanism involving AKT inhibition–dependent GSK-3β–mediated increased c-Myc and Pim-1 proteasomal degradation.

AZD1208 and quizartinib co-treatment was well tolerated in the orthotopic model.

Purpose: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition.Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo.Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor–induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML.Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234–47. ©2017 AACR.

Supplementary figure legends

Leather was an important commodity for the British empire in terms of industrial production and scientific innovation. From the mid nineteenth century in India, the British sought to convert leathe...

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.

Learning outcomes After completing the case, students should be able to understand: the importance and uniqueness of the individual market and developing a suitable marketing strategy. The concept of value creation and learn the importance of developing the right value proposition to compete and succeed in a market. The target audience and how to create the right marketing mix. Competition in a digital landscape and the importance of developing an appropriate strategy to counter its rivals and position the brand effectively. Case overview/synopsis During his visit to India in December 2019, Netflix’s founder and chief executive officer Reed Hastings talked about a series of steps the company had taken in the recent past to successfully face stiff competition and move towards achieving its stated target of 100 million viewers. These steps involved significant changes in their marketing mix such as reworking their pricing, developing a rich portfolio of Indian content and building various partnerships. Since Netflix’s launch in India (December 2016), it faced fierce competition from players such as Hotstar and Amazon Prime, both of whom had developed a rich portfolio of Indian content and adopted a very aggressive pricing strategy thus, making these changes essential. At the time of their launch, Netflix had set a very ambitious target of gaining 100 million viewers within five years (by 2021) while adopting a premium pricing strategy and positioning themselves uniquely based on their international content. They quickly learned that they would have to reevaluate their approach if they wanted to achieve their target on time. The changes announced by Hastings were an effort in that direction. The moot question was whether these steps would help Netflix India reach its goal. This challenge was further compounded by an almost 40% hike in data tariffs by three major wireless carriers considering most Indians watched over-the-top media content on their mobile phones. Complexity academic level The case is designed for undergraduates, as well as for fundamental marketing courses in the Master of Business Administration and other graduate level programmes. It can be taught in the Principles of Marketing, Marketing Strategy and International Marketing courses. It is ideal for topics such as understanding the operation of a digital business in a new market, customer value creation and value drivers, brand and brand positioning, product promotion, strategies for business growth and expansion, fighting competition in a digital landscape. Supplementary materials Teaching Notes are available for educators only. Subject code CSS 8: Marketing.

Leather is a sensuous object marked by complex affects of desire and disgust. In India, this disgust is amplified due to the association of leather with caste. This paper examines the leather tanne...

Fms-like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD–expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD–expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo. PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3β, and GSK-3β inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3β activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3β also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD–expressing cells through a novel mechanism involving AKT inhibition–dependent GSK-3β–mediated increased c-Myc and Pim-1 proteasomal degradation.

This essay examines the question of empathy within the discourses of caste in India and argues that the presence of this deeply hierarchical system, which is premised on the idea of disgust, does not allow for the production of empathy or empathic political spaces. Locating itself in a particular case of caste violence and its counter discourse in the Una District of the state of Gujarat in western India, this essay examines the affectual politics of the presence of the animal and the animal-like in caste publics and the consequences that it has for the question of empathy.

BACKGROUND Internal tandem duplication in the fms-like tyrosine kinase 3 receptor tyrosine kinase (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients, associated with poor treatment outcomes due to rapid relapse. FLT3 inhibitors are used in the clinic, but with incomplete efficacy and development of resistance. Further treatment options are needed. The serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD; it directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual targeting of Pim-1 and FLT3 is a promising treatment strategy. The c-Myc transcription factor contributes to dysregulation of cell growth and apoptosis in cancers, including AML. In addition to transcriptional regulation, c-Myc is regulated post-translationally by T58 phosphorylation by the serine/threonine kinase glycogen synthase kinase-3- β (GSK-3β). Here we show that concurrent treatment of cells with FLT3-ITD with Pim and FLT3 inhibitors activates GSK-3β, which phosphorylates and post-translationally downregulates c-Myc. METHODS Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and FLT3-ITD AML patient blasts were cultured with the pan-Pim inhibitor AZD1208 (1 μM) and/or the FLT3 inhibitors gilteritinib or quizartinib (15 nM, 1 nM), with and without the GSK-3β inhibitor TCG-24 (20 μM). c-Myc, p-GSK3-α/β (S21/9) and GSK3-α/β protein expression was measured by immunoblotting. c-Myc mRNA was measured by qPCR. Cells were also cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM) to measure protein half-life and proteasomal degradation. To study the role of c-Myc overexpression and activation, Ba/F3-ITD cells were infected with retroviral estrogen receptor (ER)-Myc plasmid, causing c-Myc nuclear translocation when activated by 4-hydroxytamoxifen (4-OHT; 300 nM). To study the role of c-Myc phosphorylation at T58, Ba/F3-ITD cells were infected with MycT58A plasmid, preventing c-Myc phosphorylation at T58. Apoptosis was detected by Annexin V and propidium iodide staining, measured by flow cytometry. RESULTS Treatment with Pim inhibitor AZD1208 and FLT3 inhibitor gilteritinib or quizartinib combination rapidly downregulated c-Myc protein expression in Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and in primary FLT3-ITD AML patient blasts, compared to quizartinib or gilteritinib alone. Pim inhibitor and FLT3 inhibitor combination treatment did not decrease c-Myc mRNA levels, but markedly decreased c-Myc protein half-life, from 36 mins without drugs and 24 mins with gilteritinib to 18 mins with combination. Half-life did not decrease when cells were pre-treated with the proteasome inhibitor MG-132, consistent with post-translational downregulation through proteasomal degradation. Apoptosis induction by Pim inhibitor and FLT3 inhibitor combination decreased by more than 50% in Ba/F3-ITD cells infected with ER-Myc plasmid and treated with 4-OHT, demonstrating the major role of c-Myc downregulation in apoptosis induction by combination treatment. GSK-3b is inactivated by phosphorylation, and combination treatment rapidly decreased p-GSK-3b levels, while total GSK-3b levels were unchanged, indicating activation of GSK-3b. Treatment of cells with FLT3-ITD with the GSK-3b inhibitor TCG-24 in addition to Pim and FLT3 inhibitors abrogated c-Myc protein downregulation, demonstrating that Pim and FLT3 inhibitor combination downregulates c-Myc through activation of GSK-3b. Finally, Pim and FLT3 inhibitor combination treatment did not downregulate c-Myc in Ba/F3-ITD cells transfected with c-Myc T58A, preventing c-Myc phosphorylation at T58, showing that c-Myc phosphorylation at T58 is necessary for its downregulation by combination treatment. CONCLUSIONS Concurrent treatment of cells with FLT3-ITD with Pim kinase inhibitor enhances the efficacy of FLT3 inhibitors through activation of GSK-3β and GSK-3β-mediated phosphorylation of c-Myc at T58, with resulting c-Myc downregulation through increased proteasomal degradation. This work and previous work in our laboratory on PP2A activating drugs and FLT3 inhibitor combination (Mol Cancer Ther 20:676, 2021) support GSK-3β activation as a mechanism for enhancing efficacy of FLT3 inhibitors in AML with FLT3-ITD. Disclosures No relevant conflicts of interest to declare.

In the present study, we report a highly effective electrochemical sensor for detecting 2,4-dinitrotoluene (2,4-DNT). The amperometric determination of 2,4-DNT was carried out using a gold electrode modified with zinc&ndash, metal organic framework-8 and silver quantum dot (Zn-MOF-8@AgQDs) composite. The synthesized nanomaterials were characterized by using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD). The synthesized nanocomposite proved to be efficient in electro-catalysis thereby reducing the 2,4-DNT. The unique combination present in Zn-MOF-8@AgQDs composite offered an excellent conductivity and large surface area enabling the fabrication of a highly sensitive (&minus, 0.238 &micro, A &micro, M&minus, 1 cm&minus, 2), selective, rapid and stable 2,4-DNT sensor. The dynamic linear range and limit of detection (LOD) was about 0.0002 &micro, M to 0.9 &micro, M and 0.041 &micro, M, respectively. A 2,4-DNT reduction was also observed during the linear sweep voltammetry (LSV) experiments with reduction peaks at &minus, 0.49 V and &minus, 0.68 V. This is an unprecedented report with metal organic framework (MOF) composite for sensing 2,4-DNT. In addition, the presence of other species such as thiourea, urea, ammonia, glucose, and ascorbic acid displayed no interference in the modified electrode suggesting its practicability in various environmental applications.

In the present study, a porous Zn-MOF@rGO (Zinc-Metal organic frameworks and reduced graphene oxide) was prepared via solvothermal technique and utilized for the amperometric determination of hydrazine. Zn-MOF@rGO solution was used to modify gold electrode (AuE) for the superior electrocatalytic oxidation of hydrazine in real water sample. The synthesized material was analyzed using XRD, TEM, FTIR, TGA and BET techniques. TEM studies revealed prismatic shape of Zn-MOF with size ranging between 160 and 180 nm and were well dispersed on the rGO sheets. Also, the incorporation of Zn-MOF into rGO leads to the significant improvement in the performance which can be ascribed to the large surface area, excellent adsorption affinity, chemical stability and high electrical conductivity. This is the first report on the use of Zn-MOF@rGO for the detection of hydrazine. The hydrazine sensor using Zn-MOF@rGO as electrocatalyst displayed a low LOD (8.7 × 10−3 μM), high sensitivity (5.4 × 10−2 μA μM−1 cm−2) and a fast response time (

Purpose: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition. Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo. Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor–induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML. Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234–47. ©2017 AACR.

Introduction: Dental anxiety is patient's psychological response to the dental environment. Local anesthetic injection is the most anxiety-causing procedure for children in dentistry, especially the inferior alveolar nerve block (IANB) which is the most commonly used injection technique for achieving local anesthesia of mandibular molars. The various alternatives explored for IANB are intra-ligamentary technique/periodontal injection technique and buccal infiltration. Aim: The aim of this study was to compare the effectiveness of articaine hydrochloride 4% with adrenaline 1:100,000 for the extraction of mandibular primary molar teeth using buccal infiltration injection and intraligamentary injection. Materials and Methods: The present study was conducted on 30 children in the age group of 5–10 years requiring bilateral extraction of primary mandibular molars. The patients were randomly selected and divided into two groups. Group 1 received anesthesia using buccal infiltration injection technique and Group 2 received anesthesia using intraligamentary injection both containing 4% articaine with 1:1,00,000 adrenaline. Results: The success rate of buccal infiltration and intraligamentary injection using 4% articaine with mean sound, eye, and motor scores (± standard deviation) was found to be statistically nonsignificant, i.e., 3.07 for buccal infiltration and 2.97 for intraligamentary injection technique with P = 0.835 (P > 0.05). Conclusion: There is no significant difference between buccal infiltration technique and intraligamentary injection technique for the extraction of primary mandibular molars using 4% articaine with 1:1,00,000 adrenaline.

Introduction fms-like tyrosine like kinase 3 internal tandem duplication (FLT3-ITD), present in acute myeloid leukemia (AML) cells of 30% of patients, results in constitutive and aberrant FLT3 signaling and, clinically, short disease-free survival. Efficacy of FLT3 inhibitors is limited and transient, but may be enhanced by dual targeting of FLT3-ITD signaling pathways. The tumor suppressor protein phosphatase 2A (PP2A) is inhibited in cells with FLT3-ITD. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated and also stabilized by PP2A inhibition in cells with FLT3-ITD. Pim-1 contributes directly to FLT3-ITD proliferative and anti-apoptotic effects, and also phosphorylates and stabilizes FLT3-ITD in a positive feedback loop. Moreover FLT3-ITD, PP2A and Pim-1 all regulate the transcription factor c-Myc. PP2A-activating drugs enhance efficacy of FLT3 inhibitors. We sought to identify mechanisms underlying the efficacy of this combination. Methods Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and blasts from patients with AML with FLT3-ITD were cultured with the FLT3 inhibitors gilteritinib (15 nM) or quizartinib (1 nM) and/or the PP2A-activating drugs FTY720 (2-4 µM) or DT-061 (10 µM), or with DMSO control. Pim-1, c-Myc, p-AKT (S473 and T308) and AKT protein expression was measured by immunoblotting, along with p-STAT5 (Y694), STAT5, p-PP2A (Y307) and PP2A expression. To study post-translational regulation, cells were cultured with cycloheximide (100 µg/mL) with and without the proteasome inhibitor MG-132 (20 µM). Ubiquitinated c-Myc was measured by co-immunoprecipitation and immunoblotting with c-Myc and ubiquitin antibodies. Ba/F3-ITD cells were stably transfected with estrogen receptor (ER)-c-Myc, kinase-dead Pim-1 or myristoylated AKT plasmids or corresponding empty vectors. Apoptosis was detected by Annexin V and propidium iodine staining, measured by flow cytometry. Cells were also cultured with the pan-Pim kinase inhibitor AZD1208 (1 µM), the Myc inhibitor 10058-F4 (100 µM) or the pan-AKT inhibitor MK-2206 (5 µM). Results Concurrent treatment of Ba/F3-ITD and MV4-11 cells and primary AML cells with FLT3-ITD with a FLT3 inhibitor (gilteritinib or quizartinib) and a PP2A-activating drug (FTY720 or DT-061) decreased growth and increased apoptosis induction, relative to treatment with single drugs. Concurrent FLT3 inhibitor and PP2A-activating drug treatment decreased expression of both Pim-1 and c-Myc protein. Concurrent treatment decreased Pim-1 half-life from 15 to 5 minutes, and c-Myc half-life from 30 to 5 minutes, while half-lives were restored by concurrent treatment with the proteasome inhibitor MG-132. Concurrent treatment was also shown to increase c-Myc ubiquitination. Effects of concurrent treatment on Pim-1 and c-Myc were independent, as transfection with kinase-dead Pim-1 or treatment with Pim inhibitor AZD1208 did not alter c-Myc downregulation, and c-Myc overexpression or treatment with Myc inhibitor 10058-F4 did not alter Pim-1 downregulation. Concurrent treatment with FLT3 inhibitor and PP2A-activating drug did not alter expression of c-Myc deubiquitinases, but rapidly decreased AKT S473 and T308 phosphorylation. FLT3 inhibitor and PP2A activator co-treatment did not induce downregulation or increased turnover of Pim-1 and c-Myc protein or apoptosis in cells with constitutive AKT activation caused by transfection of myristoylated AKT. Moreover, AKT inhibition downregulated Pim-1 and c-Myc protein expression, decreased Pim-1 and c-Myc protein half-lives from 15 to 5 minutes and 30 to 10 minutes, respectively, and induced apoptosis of cells with FLT3-ITD, replicating the effects of FLT3 inhibitor and PP2A activator co-treatment. Conclusion PP2A activators enhance the efficacy of FLT3 inhibitors in AML cells with FLT3-ITD through AKT inactivation-dependent increased Pim-1 and c-Myc proteasomal degradation, which is a novel mechanism. The data support further preclinical and clinical testing of this dual targeting approach to treatment of AML with FLT3-ITD. Disclosures Baer: Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

Introduction: In 30% of acute myeloid leukemia (AML) patients, internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) causes constitutive and aberrant FLT3 signaling, and these patients have short relapse-free and overall survival. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. FLT3 activation also inhibits the tumor suppressor protein phosphatase 2A (PP2A). FLT3 inhibitors and PP2A-activating drugs have been shown to induce synergistic cytotoxicity in cells with FLT3-ITD. To address mechanisms underlying this effect, we studied effects of combination therapy on the oncogenic serine/threonine kinase Pim-1 and the transcription factor c-Myc, both of which are upregulated in cells with FLT3-ITD and are also PP2A substrates. Methods: Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and/or the PP2A-activating drug fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Drug combination effects were measured by combination index determined by the Chou-Talalay method using CompuSyn software. Apoptosis was measured by Annexin V/propidium iodide staining detected by flow cytometry. c-Myc and GAPDH control mRNA was measured by real-time polymerase chain reaction. Pim-1 kinase, c-Myc, phospho-c-MycSer62, phospho-c-MycThr58, phospho-STAT5Tyr694, STAT5, phospho-PP2ATyr307, PP2A, phospho-BADSer112 and BAD levels were measured by immunoblotting. Cycloheximide treatment was used to assess protein stability. Protein expression and stability were measured with and without the proteasome inhibitor MG-132. Pim-1 kinase was inhibited with the pan-Pim inhibitor AZD1208. Ba/F3-ITD cells were infected with pMX-Flag-K67M kinase-dead (KD) Pim-1 and empty pMX retroviral vectors and with pBABE-ER-cMYC and with empty pBABE-ER retroviral vectors. Results: Concurrent treatment with 15 nM gilteritinib or 1 nM quizartinib and FTY720 2 µM in cell lines and 4 µM in patient samples decreased growth and increased apoptosis of cells with FLT3-ITD, relative to single drug treatments, and produced synergistic cytotoxicity. FLT3 inhibition was confirmed by decrease in phospho-STAT5 and PP2A activation by decreased phospho-PP2A. Concurrent treatment decreased expression of both Pim-1 and c-Myc protein, but not c-Myc mRNA, in Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD, relative to single drug treatments. Additionally, selective decrease in phospho-MycSer62, a stable c-Myc phosphoprotein that is dephosphorylated by PP2A, was seen, with persistence of phospho-c-MycThr58. FLT3 inhibitor and PP2A activator combination treatment was found to decrease stability of c-Myc and Pim-1 protein, in relation to single drugs. Moreover, pretreatment with the proteasome inhibitor MG-132 abrogated downregulation of Pim-1 and c-Myc protein expression and decrease in Pim-1 and c-Myc protein stability in Ba/F3-ITD cells treated with FLT3 inhibitor and PP2A activator. Pretreatment with the pan-Pim kinase inhibitor AZD1208, with Pim-1 inhibition confirmed by decreased phospho-BADS112 had no effect on c-Myc downregulation, and c-Myc was similarly downregulated in Pim-1 kinase-dead cells as in parental and empty-vector cells, demonstrating that combination treatment effects on c-Myc are not Pim-1 kinase-dependent. Additionally, FLT3 inhibitor and PP2A-activating drug combination induced apoptosis in 30% of cells with c-Myc overexpression, compared to 60% of parental and empty vector-infected cells. Finally, c-Myc overexpression did not abrogate Pim-1 downregulation by combination treatment. Conclusions: Concurrent FLT3 inhibitor and PP2A activating drug treatment induces synergistic cytotoxicity in AML cells with FLT3 internal tandem duplication through proteasomal degradation of Pim-1 and c-Myc, and effects on Pim-1 and c-Myc are independent. The data support in vivo testing of FLT3 inhibitor and PP2A-activating drug combinations and development of a clinical trial. Disclosures No relevant conflicts of interest to declare.

Background: In 30% of acute myeloid leukemia (AML) patients, the presence of fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) causes constitutive FLT3 signaling, and these patients relapse rapidly following response to chemotherapy. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to its proliferative and antiapoptotic effects and also phosphorylates and stabilizes it in a positive feedback loop. FLT3 activation also inhibits the tumor-suppressor protein phosphatase 2A (PP2A), causing stabilization of Pim-1 protein. FLT3 inhibitors and PP2A-activating drugs (PADs) are known to synergistically induce cytotoxicity in cells with FLT3-ITD, and here we studied mechanisms underlying this effect. Methods: Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 cells, with FLT3-ITD, were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and the PAD fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Apoptosis was measured by Annexin V/propidium iodide staining. Drug combination indexes were determined by the Chou-Talalay method with CompuSyn software. Pim-1 kinase, Myc, MycSer62, MycThr58, STAT5, phospho-STAT5, PP2A and phospho-PP2A levels were measured by immunoblotting. Cycloheximide was used to assess protein stability. Results: Concurrent treatment with ASP2215 or AC220 and FTY720 decreased growth and enhanced apoptosis of cells with FLT3-ITD, relative to single drugs, and produced synergistic cytotoxicity. Concurrent treatment sequentially decreased expression of the 44 kDa Pim-1 isoform (Pim-1L) and then c-Myc, via decreased Pim-1L and c-Myc protein stability. Co-incubation with the proteasome inhibitor MG-132 prevented drug-induced degradation and restored Pim-1L and Myc expression in cells co-treated with FLT3 inhibitor and PAD. Drug-induced Myc destabilization/proteasomal degradation was associated with decreased MycSer62 and increased MycThr58 levels. Treatment with the pan-Pim kinase inhibitor AZD1208 prior to ASP2215 or AC220 and FTY720 treatment markedly increased MycThr58 levels but did not affect MycSer62, consistent with both Pim-dependent and -independent effects of FLT3 inhibition and PP2A activation on c-Myc expression. Conclusions: Combined FLT3 inhibitor and PAD treatment synergistically induces cytotoxicity in cells with FLT3-ITD by enhancing Pim-1 and Myc proteasomal degradation, and effect on Myc is both Pim-1-dependent and -independent. The data support in vivo testing of FLT3 inhibitor and PAD combinations. Citation Format: Mario Scarpa, Shivani Kapoor, Sikemi Ibikunle, Andre Rush, Patrick R. Baldwin, Danilo Perrotti, Maria R. Baer. FLT3 inhibitors and PP2A-activating drugs synergistically induce cytotoxicity in AML cells with FLT3-ITD through enhanced Pim-1 and Myc proteasomal degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 834.

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival following both chemotherapy and allogeneic hematopoietic stem cell transplantation. Inhibitors of FLT3 signaling have shown activity in clinical trials in FLT3-ITD AML, but efficacy has generally been limited and transient. Concurrent inhibition of other targets in FLT3-ITD signaling pathways is being explored as an approach to increasing the depth and duration of responses to FLT3 inhibitors. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3, thereby promoting FLT3 signaling in a positive feedback loop in cells with FLT3-ITD. Pim kinase inhibitors are in clinical trials. We previously showed that combinations of clinically active Pim kinase and FLT3 inhibitors at pharmacologically relevant concentrations enhance apoptosis and decrease clonogenic growth of FLT3-ITD AML cell lines and primary patient cells in vitro and suppress growth of FLT3-ITD AML cells in vivo, in relation to treatment with FLT3 or Pim inhibitors alone. Here we studied the mechanistic effects of concurrent Pim kinase and FLT3 inhibition, demonstrating a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells. Ba/F3-ITD cells, transfected with FLT3-ITD, were cultured with the pan-Pim kinase inhibitor AZD1208 at 1 μM, a concentration chosen based on in vitro and phase I clinical trial data, and/or the FLT3 inhibitor quizartinib at 1 nM, its IC50 concentration, and expression of the anti-apoptotic proteins Mcl-1, Bcl2 and Bcl-xL and the pro-apoptotic proteins BAD/S112 p-BAD, BAK, BAX and Bim was measured by western blot analysis. Mcl-1 expression decreased in a time-dependent manner with AZD1208 and quizartinib co-treatment, but not with treatment with either inhibitor alone, while levels of the other proteins did not change. Mcl-1 downregulation with Pim kinase and FLT3 inhibitor combination treatment was then confirmed in the human FLT3-ITD AML cell lines MV4-11 and MOLM-14. Mcl-1 expression is regulated at multiple levels, and we next sought to determine the mechanism(s) by which it is downregulated by concurrent Pim and FLT3 inhibition. While Mcl-1 protein levels decreased, Mcl-1 mRNA levels did not change, indicating post-transcriptional regulation. Additionally, levels of miR-29b, a negative regulator of Mcl-1 translation,decreased similarly in Ba/F3-ITD cells treated with AZD1208 and quizartinib, compared to quizartinib alone. Polysome profiling showed decreased total mRNA translation, but no selective reduction in Mcl-1 translation. In contrast, the progressive decrease in Mcl-1 protein expression with AZD1208 and quizartinib co-treatment was abrogated by addition of the proteasome inhibitor MG-132, demonstrating that Mcl-1 protein is downregulated by enhanced Mcl-1 proteasomal degradation. This mechanism was further confirmed by demonstration of an increase in ubiquitinated Mcl-1 prior to Mcl-1 downregulation in cells co-treated with AZD1208 and quizartinib, but not with each inhibitor alone or with DMSO control. The deubiquitinase USP9X decreases Mcl-1 ubiquitination and consequent proteasomal degradation, and we found that USP9X expression is downregulated prior to the increase in ubiquitinated Mcl-1 and the subsequent decrease in Mcl-1 protein levels during AZD1208 and quizartinib co-treatment, but was not altered by treatment with either inhibitor alone. In contrast, expression of the ubiquitin E3 ligases Mule/ARF-BP1, SCFβ-TrCP and Trim17, which mediate Mcl ubiquitination, did not change prior to Mcl-1 downregulation. Preclinical studies in our laboratory and others have shown in vitro and in vivo efficacy of combination treatment with Pim kinase and FLT3 inhibitors in FLT3-ITD AML, suggesting clinical promise of this approach. Here we show that, mechanistically, concurrent Pim kinase and FLT3 inhibition causes a post-translational decrease in expression of the anti-apoptotic protein Mcl-1 via enhanced proteasomal degradation, preceded by downregulation of the Mcl-1 deubiquitinase USP9X and an increase in ubiquitinated Mcl-1, a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells. Disclosures Tron: AstraZeneca: Employment; AstraZeneca: Employment. Huszar:AstraZeneca: Employment.

Introduction: Internal tandem duplication of the fms-like tyrosine kinase-3 receptor (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients and these patients have short disease-free survival following chemotherapy. FLT3 inhibitors are clinically active, but their activity is limited and transient. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, and promotes FLT3 signaling in a positive feedback loop in FLT3-ITD cells. We have previously demonstrated that inhibiting Pim kinase sensitizes AML cell lines and primary AML patient blasts with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitor chemotherapy drugs. Here we studied the effects of the pan-Pim kinase inhibitor PIM447 (formerly LGH447; Novartis Pharmaceuticals), currently in clinical trials, on response of FLT3-ITD-expressing cell lines and AML patient samples to FLT3 inhibitors and to topoisomerase 2 inhibitors, with the ultimate goal of developing clinically applicable combination regimens. Methods: Cell lines and AML patient samples with FLT3-ITD were cultured with FLT3 inhibitors or topoisomerase 2 inhibitors at clinically applicable concentrations and PIM447 at a range of concentrations. Apoptosis was measured by Annexin V labeling and PARP cleavage. Mcl-1 expression was measured by immunoblotting. Reactive oxygen species (ROS) were measured with the redox-sensitive dye CM-H2DCFDA and DNA double-strand breaks (DSBs) by immunoblotting for γH2AX. Results: Transfected Ba/F3-ITD and 32D/ITD cells, MV4-11 and MOLM-14 human AML cells and primary AML patient blasts, all expressing FLT3-ITD, were treated with FLT3 inhibitors, including 100 nM midostaurin and 1 nM quizartinib, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 produced a concentration-dependent increase in apoptosis induced by each FLT3 inhibitor (p Conclusions: The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes AML cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. Our data support in vivo testing of combination regimens and design of clinical trials aimed at improving outcomes for patients with AML with FLT3-ITD. Citation Format: Kshama A. Doshi, Patrick R. Baldwin, Shivani Kapoor, Maria R. Baer. The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes acute myeloid leukemia cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3866.

The future of any organization depends on the strategic use of their human resources irrespective of the size and domain of the industry. The research in this field is new and focuses on the importance of Strategic Human Resource Management (SHRM) in today's competitive work environment. The paper in an attempt to review literature, discusses various definitions that have evolved in SHRM over a period of time. This review work shows that there is diversity over the meaning of Strategic Human Resource Management Practice in the literature.