BaF3 Cells Research Articles

Abstract 5199: JNJ-61186372, an EGFR-cMet bispecific antibody, in EGFR Exon 20 insertion-driven advanced non-small cell lung cancer (NSCLC)

Abstract PURPOSE: Although EGFR exon20 insertion (ex20ins) mutations account for 4~12 % of EGFR mutant NSCLC patients, there is no effective and selectable anticancer drugs targeting ex20ins mutations so far due to various variant mutations in ex20ins mutations. Moreover, most EGFR ex20ins mutants show primary resistance to EGFR TKIs. JNJ-61186372 (JNJ-372), a bispecific antibody that targets the EGFR and cMet receptors, is currently being explored in a first-in-human study in patients with NSCLC. To better understand the mechanism of JNJ-372 activity in this patient population, we conducted preclinical studies exploring the activity of JNJ-372 in different EGFR ex20ins models. METHODS: To elucidate whether JNJ-372 has antitumor effect in EGFR ex20ins mutants via EGFR and c-MET inhibition, cell viability, western blot, cell cycle, colony formation assay, FACS analysis were performed in JNJ-372 treated BaF3 cells, PDCs, PDOs, and PDX expressing EGFR ex20ins mutation. For mouse tumor models, JNJ-372 was administered i.p. twice a week at 10 mg/kg or 30 mg/kg. Antibody dependent cellular cytotoxicity (ADCC) assay was assessed to figure out whether JNJ-372 had ADCC effects. Referenced patients with ex20ins disease were administered 1050 mg JNJ-372 i.v. weekly for the first 4-week cycle, then biweekly for each subsequent cycle RESULTS: JNJ-372 inhibited the growth of BaF3 cells, PDCs, and a PDO harboring a range of ex20ins, which were resistant to osimertinib and gefitinib. Mechanistic assays revealed the reduction of EGFR and cMet receptor levels and decreases in phospho-EGFR and c-Met, as well as inhibition of their downstream signaling pathways. Cleaved caspase-3 and BIMEL were upregulated at anti-proliferative doses, suggesting caspase-mediated cell death. JNJ-372 demonstrated corresponding antitumor activity in PDC and PDX models harboring different ex20ins; inhibition of signaling and engagement of the apoptotic pathway was confirmed in tumors of JNJ-372-treated mice. In PDCs with EGFR ex20ins mutation, we verified that JNJ-372 had a significant ADCC effect compared to EGFR antibody drug cetuximab. In the first-in-human trial, CT scans from two patients treated with JNJ-372 revealed reductions in tumor burden. A 58-year patient harboring H773delinsNPY showed -63% tumor reduction with progression-free survival of > 20 months and a 48-year patient harboring S768_D770dup showed -38.9% tumor reduction. CONCLUSION: JNJ-372 drives antitumor activity in preclinical models of EGFR ex20ins, which have no therapeutic options in clinic, by decreasing EGFR and cMet receptor levels, inhibiting downstream signaling cascades, activating apoptotic signaling as well as ADCC. These results provide a promising therapeutic option to patients with EGFR ex20ins mutations and an understanding of the activity of JNJ-372 being observed in the first-in-human study. Citation Format: Jiyeon Yun, Han Na Kang, Soo-Hwan Lee, Seo-Yoon Jeong, Chae Won Park, Jae-Hwan Kim, Kyoung-Ho Pyo, Ji Min Lee, Seok-Young Kim, Min Hee Hong, Hye Ryun Kim, Meena Thayu, Joshua Curtin, Roland Knoblauch, Matthew Lorenzi, Byoung Chul Cho. JNJ-61186372, an EGFR-cMet bispecific antibody, in EGFR Exon 20 insertion-driven advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5199.

Abstract 3087: FCN-098, a novel second-generation TRK inhibitor, overcomes TRK resistance mutations in vitro and in vivo

Abstract Tropomyosin receptor kinases (TRK or NTRK) are the transmembrane tyrosine kinases activated by soluble growth factors named neurotrophins (NT). TRK family includes three different members, namely TRKA, TRKB and TRKC. Extracellular recognition of NT to TRK proteins induces receptor dimerization, phosphorylation, and activation of the downstream signal transduction pathways via PI3K, RAS/MAPK/ERK, and PLC-gamma. TRKs are expressed primarily in neuronal tissues and regulate differentiation and survival of neuronal cells. Alteration of TRK pathway caused by gene fusion of TRK is frequently associated with the poor prognosis of a wide range of solid malignancies, such as neuroblastoma, breast cancer, pancreatic cancer, melanoma, thyroid cancer, glioblastoma, colorectal cancer, sarcomas, cholangiocarcinoma, and non-small cell lung cancer. The efficacy of TRK inhibition in TRK fusion-positive cancers has been demonstrated by the high response rate and lasting effect of the first-generation TRK inhibitors larotrectinib and entrectinib. However, acquired resistance mutations of TRK, such as solvent front mutations, gatekeeper mutations and DFG motif mutations, often emerge and abrogate the inhibitory effect of first-generation inhibitors. Thus, developing novel TRK inhibitors that can effectively overcome resistance mutations can provide great clinical benefits to patients. Here we introduce FCN-098, a potent second-generation TRK inhibitor. FCN-098 demonstrated marked kinase inhibition activity against wild-type (WT) and mutant TRK with IC50 at sub-nano or single nanomolar range. FCN-098 significantly and dose-dependently inhibited phosphorylation of TRK and its downstream effector ERK in TRK fusion-positive KM12 human colon cancer cells and BaF3 cells ectopically overexpressing WT or mutant TRK fusions. FCN-098 potently inhibited proliferation of KM12 cells and BaF3 cells overexpressing WT or various mutant TRK fusions while sparing TRK-negative cell lines. Consistent with in vitro results, FCN-098 showed remarkable anti-tumor activity in xenograft models driven by WT or mutant TRK fusions in a dose-dependent manner. In particular, the anti-tumor activity of FCN-098 was superior to another second-generation TRK inhibitor LOXO-195 at the same dose in xenografts driven by TPM3-TRKA G595R or ETV6-TRKC G623R mutation, and exhibited comparable potency to LOXO-195 in LMNA-TRKA G667C-driven xenografts. In non-clinical studies, FCN-098 exhibited excellent pharmacokinetic (PK) and safety properties, demonstrating longer T1/2 in both dogs and rats along with higher Cmax and larger dose-normalized AUC in dogs compared to LOXO-195. Overall, FCN-098 overcomes TRK resistance mutations in vitro and in vivo and exhibits favorable PK and safety profiles. FCN-098 has the potential to become an effective and safe therapeutic choice for a wide range of solid tumors harboring WT or mutant TRK fusions. Citation Format: Shu Lin, Tongshuang Li, Xianlong Wang, Lijun Yang, Zhifang Chen, Yue Rong, Xingdong Zhao, Yanxin Liu, Zongyao Zou, Min Lin, Yuwei Gao, Jiashu Zhou, Weibo Wang. FCN-098, a novel second-generation TRK inhibitor, overcomes TRK resistance mutations in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3087.

Abstract LB-109: Discovery of a highly potent and blood-brain barrier (BBB) penetrable second generation Pan-TRK/ROS1 dual inhibitor, XZP-5955

Abstract Oncogenic TRK fusions are identified in various types of cancers including brain cancers in adults and children. Currently in clinic, first generation TRK inhibitors, such as larotrectinib and entrectinib, have demonstrated marked efficacy in patients with cancers carrying TRK fusions, however, like most kinase inhibitors, acquired resistance mediated by kinase domain mutations has occurred. To overcome the acquired resistances, second-generation TRK inhibitors, TPX-0005 and LOXO-195, targeting both wild-type and mutant TRK fusions are currently in clinical development. Both TPX-0005 and LOXO-195 have demonstrated efficacies in both pre-clinical animal models and clinical studies in patients. The potential shortcoming for both TRK inhibitors of TPX-0005 and LOXO-195 is their limited ability to penetrate blood-brain barrier (BBB) in order to effectively target TRK fusion related tumors inside brain. Here we report a highly potent and orally bioavailable pan-TRK/ROS1 dual inhibitor, XZP-5955. XZP-5955 shows high potency against a broad spectrum of TRK and ROS1 resistance mutations found in patients including TRKA G595R (IC50=2.2nM), TRKA G667C (IC50=1.0nM), TRKC G623R (IC50=4.7nM) and ROS1 G2032R (IC50 < 0.05 nM). It also potently inhibits proliferations of rat BaF3 cells expressing oncogenic NTRK or ROS1 fusions and their resistant variants, such as TRKA G595R, TRKC G623R, TRKA G667C and ROS1 G2032R with high potency. XZP-5955 demonstrated superior potency in biochemical and cell assays to both TPX-0005 and LOXO-195. In in vivo, XZP-5955 is highly effıcacious against NTRK mutant cell line in xenograft models, and it is well tolerated in mice after repeated oral administration. In a BaF3 LMNA-TRKA G595R xenograft model, oral dosing at 1 mg/kg, 3 mg/kg and 10 mg/kg for 9 days, XZP-5955 demonstrated dose dependent inhibition of tumor growth and led to significant tumor regression or even complete tumor inhibition at 3mg/kg. In addition, XZP-5955 has good PK profile in multiple species. Noticeably, XZP-5955 demonstrates excellent brain penetration in rodent species, such as with brain/plasma ratio of 2.2 in rat. This is in contrast to the poor brain penetration observed with the other relevant inhibitors targeting TRKs. Preliminary toxicology studies in rat and dog demonstrated that XZP-5955 has desired margins of safety to warrant its further development. Based on the activity profile of XZP-5955 against clinically-relevant resistance mutations and its excellent brain penetration, this molecule has high potential to become a second-generation TRK kinase inhibitor especially for targeting brain tumors or brain metastatic lesions that harbor a NTRK gene fusion and ROS1 mutations. XZP-5955 is currently undergoing further characterizations in IND enabling studies. IND submission is expected by the end of 2020. Citation Format: Jun Sun, Shujin Bai, Bo Chen, Yuqi Dong, Yanwei Ding, Qian Hu, Xifeng Ma, Yanfang Nie, Chunmin Shi, Ying Sun, Zhaoqiang Wei, Xiping Yang, Peng Zhu, Chengkong Shi, Jiakui Li, Chongtie Shi, Zuliang Yao, Bin Liu. Discovery of a highly potent and blood-brain barrier (BBB) penetrable second generation Pan-TRK/ROS1 dual inhibitor, XZP-5955 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-109.

Abstract 5198: JNJ-61186372, a novel EGFR/c-Met bispecific antibody, exhibits potent antitumor activity in broad-spectrum of acquired resistance to EGFR-TKIs

Abstract Background: 1st generation (erlotinib & gefitinib), 2nd generation (afatinib), and 3rd generation (osimertinib) EGFR TKIs have marked efficacy in patient with EGFR-mutant NSCLC. Unfortunately, patients who show T790M (-) mutation after treated with 1st and 2nd generation EGFR TKI or have C797S (+) mutation or other mechanisms of acquired resistance to 3rd generation EGFR TKI have no therapeutic options for curing cancer in clinic. We introduce a potent therapeutics for overcoming acquired resistance to EGFR-TKIs in several EGFR mutant NSCLC models using JNJ-61186372 (JNJ-372), a bispecific antibody that targets the EGFR and c-Met receptors. Methods: We classified PDC and PDX models into two groups: i) 1st and 2nd generation EGFR TKI acquired resistance models with T790M (-) mutation (YU-1093, YU-1152), ii) 3rd generation EGFR TKI acquired resistance models with C797S (+) mutation (YU-1097), and C797S (-) mutation with unknown resistance mechanisms (YU-1095, YU-1089, YU-1096, YUX-1024_YH1R, YHIM-1035, YHIM-1045, and YHIM-1053). We established 7 patient-derived cells (PDC) and 3 patient-derived xenografts (PDX) model from patients with broad-spectrum acquired resistance to EGFR-TKIs. We performed cell viability test and western blot to investigate antitumor activity of JNJ-372. We evaluated then in vivo antitumor activity of JNJ-372 in PDX and PDC-xenograft models. JNJ-372 was administrated i.p. twice a week at 30 mg/kg. Results: We assessed the ability of JNJ-372 to overcome broad-spectrum of EGFR-TKI acquired resistances. JNJ-372 significantly inhibited cancer cell growth in vitro and in vivo in PDC model harboring T790M (-) mutation. JNJ-372 significantly decreased cell proliferation rate and EGFR downstream signaling in BaF3 cell harboring Del19 (L858R)/T790M/C797S mutant and PDC model harboring Del19/T790M/C797S mutant with amplified KRAS and loss of Rb. C797S (-) cases of four PDCs and three PDX models had i) loss of T790M with c-Met amplification or other acquired resistance mutation and ii) T790M (+) with amplified c-Met. In these models we identified dramatic tumor regression and reduction of EGFR and c-Met expression in vitro and in vivo PDX and PDC-xenograft models. Interestingly, we observed combination treatment of JNJ-372 and c-Met inhibitor showed synergistic immune cell mediated cellular cytotoxicity in EGFR-TKI acquired resistance models harboring c-Met amplification. JNJ-372 exhibited potent inhibition of tumor growth in preclinical models through targeting EGFR and c-Met receptor, decreasing EGFR and c-Met receptor level, inhibiting EGFR downstream signaling cascade and activating ADCC effect in vivo. Conclusion: Our results suggest that JNJ-372 will provide potential and effective therapeutic options for patients with no therapeutic option to overcome broad-spectrum of acquired resistance to EGFR-TKIs. Citation Format: SooHwan Lee, Jiyeon Yun, Seo-Yoon Jeong, Chae Won Park, Seok-Young Kim, Min Hee Hong, Hye Ryun Kim, Byoung Chul Cho. JNJ-61186372, a novel EGFR/c-Met bispecific antibody, exhibits potent antitumor activity in broad-spectrum of acquired resistance to EGFR-TKIs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5198.

Design, synthesis, and Structure–Activity Relationships (SAR) of 3-vinylindazole derivatives as new selective tropomyosin receptor kinases (Trk) inhibitors

Neurotrophic receptor tyrosine kinase (NTRK) fusions are oncogenic drivers for a variety of adult and pediatric tumors, validated by the US FDA approval of small molecular Trk inhibitors Larotrectinib (1, LOXO-101) and Entrectinib (2). However, gene mutation mediated resistance becomes a major challenge for Trk inhibitor therapies. Herein, we report the design, synthesis and Structure–Activity Relationship investigation of a series of 3-vinylindazole derivatives as new Trk inhibitors with low nanomolar potencies. A representative compound, 7mb, binds to TrkA/B/C with Kd values of 1.6, 3.1 and 4.9 nM, and suppresses their kinase functions with IC50 values of 1.6, 2.9 and 2.0 nM, respectively, but is obviously less potent for the majority of a panel of 403 wild-type kinases in a KINOMEscan selectivity investigation. The compound also potently suppresses proliferation of a panel of BaF3 cells stably transformed with NTRK fusions with IC50 values in low nM ranges. Additionally, the compound exhibits strong inhibition against the Larotrectinib-resistant cells with NTRK1-G667C or NTRK3-G696A mutations with IC50 values of 0.031 and 0.018 μM, respectively. Although the relatively poor oral bioavailability of 7mb will limit its further development, this compound may be utilized a lead molecule for further structural optimization.

A Small Molecule Inhibitor, OGP46, Is Effective against Imatinib-Resistant BCR-ABL Mutations via the BCR-ABL/JAK-STAT Pathway

Chronic myeloid leukemia (CML) is caused by the Philadelphia (Ph+) chromosome carrying the BCR-ABL oncogene, a constitutively active tyrosine kinase. The discovery of imatinib represents a major success story in the treatment against CML. However, mutations in the BCR-ABL kinase domain are a major cause of resistance to imatinib, demonstrating that BCR-ABL remains a critical drug target. Here, we investigate a novel small molecule inhibitor, OGP46, for its inhibitory activity against K562, a panel of murine BaF3 cell lines stably expressing either wild-type BCR-ABL or its mutant forms, including T315I. OGP46 exhibits potent activity against imatinib-resistant BCR-ABL mutations, including T315I. OGP46 induced cell differentiation accompanied by G0/G1 cell-cycle arrest and suppressed the colony formation capacity of cells. Treatment with OGP46 significantly decreased the mRNA and protein expression of BCR-ABL in K562 and BaF3-p210-T315I cells. Mechanistically, the anti-cancer activity of OGP46 induced by cell differentiation is likely through the BCR-ABL/JAK-STAT pathway in native BCR-ABL and mutant BCR-ABL, including T315I, of CML cells. Our findings highlight that OGP46 is active against not only native BCR-ABL but also 11 clinically relevant BCR-ABL mutations, including T315I mutation, which are resistant to imatinib. Thus, OGP46 may be a novel strategy for overcoming imatinib-resistance BCR-ABL mutations, including T315I.

Development and validation of targeted treatments for the rare tumor syndrome infantile myofibromatosis.

e22519 Background: Infantile myofibromatosis (IMF; MIM 228550) is an autosomal dominant disorder characterized by fibroblastic tumors of skin, muscles, viscera and bones. Tumor growth may begin during fetal development, where it may lead to early death, and/or can be present throughout adult life. We originally demonstrated that mutations in the platelet-derived growth factor receptor beta gene (PDGFRb) result in IMF. Currently, beyond surgical removal and chemotherapy, there is no effective treatment. Using an integrated genomic, bioinformatic and functional approach based on analysis of patient-derived IMF cells and mouse models, encapsulating multiple IMF-causing PDGFRb mutations, we sought to identify potentially druggable targets and validate them in preclinical models. Methods: Patient samples were genetically characterized by whole exome (WES) and RNA sequencing. Patient-derived cell lines from tumor and adjacent normal tissue (PDCL) were generated from a patient with a novel F864L mutation. This IMF-causing mutation along with two others, R561C and P660T, were stably expressed in BAF3 cells for functional characterization. PDCL-F864L were used to generate an orthotopic mouse IMF model. Results: WES of paired patient tumor, PDCL and germline samples confirmed a novel germline PDGFRb mutation (c.2590T > C, Phe864Leu). Using PDCL, the F864L mutation was shown to result in constitutive PDGFRb activation. Based on these findings, the tyrosine kinase inhibitors Imatinib and Sunitinib were chosen as candidate therapies. As predicted, they markedly inhibited cell proliferation and blocked PDGFRb activation and downstream signaling molecules AKT and ERK. Similar results were obtained in F864L, R561C and P660T mutant expressing BAF3 (natively PDGFRb null) cells when compared to WT PDGFRb expressing controls. Having established these findings using cultured cells, the effects of Imatinib and Sunitinib were next tested on an orthotopic mouse model. Given to IMF tumor mice as a single drug, both drugs markedly and sustainably decreased tumor size compared to control mice. Conclusions: Our findings demonstrate that IMF-associated PDGFRb mutations are gain-of-function mutations. Using patient-derived IMF models, Imatinib and Sunitinib effectively target these GOF mutations through PDGFRb signaling pathway inhibition. These findings provide preclinical data for targeted treatment of this rare cancer.

Discovery of triazolo [1,5-a] pyridine derivatives as novel JAK1/2 inhibitors

Small molecule JAK inhibitors have been demonstrated efficacy in rheumatoid arthritis, inflammatory bowel disease, and psoriasis with the approval of several drugs. Aiming to develop potent JAK1/2 inhibitors, two series of triazolo [1,5-a] pyridine derivatives were designed and synthesized by various strategies. The pharmacological results identified the optimized compounds J-4 and J-6, which exerted high potency against JAK1/2, and selectivity over JAK3 in enzyme assays. Furthermore, J-4 and J-6 effectively suppressed proliferation of JAK1/2 high-expression BaF3 cells accompanied with acceptable metabolic stability in liver microsomes. Therefore, J-4 and J-6 might serve as promising JAK1/2 inhibitors for further investigation.

Preclinical development of a novel BCR-ABL T315I inhibitor against chronic myeloid leukemia.

Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm primarily due to the presence of the BCR-ABL fusion gene that produces the constitutively active protein, BCR-ABL. Imatinib, a BCR-ABL-targeted drug, is a first-line drug for the treatment of CML. Resistance to imatinib occurs as a result of mutations in the BCR-ABL kinase domains. In this study, we evaluated S116836, a novel BCR-ABL inhibitor, for its anti-cancer efficacy in the wild-type (WT) and T315I mutant BCR-ABL. S116836 was efficacious in BaF3 cells with WT or T315I mutated BCR-ABL genotypes. S116836 inhibits the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. Mechanistically, S116836 arrests the cells in the G0/G1 phase of cell cycle, induces apoptosis, increases ROS production, and decreases GSH production in BaF3/WT and BaF3/T315I cells. Moreover, in mouse tumor xenografts, S116836 significantly inhibits the growth and volume of tumors expressing the WT or T315I mutant BCR-ABL without causing significant cardiotoxicity. Overall, our results indicate that S116836 significantly inhibits the imatinib-resistant T315I BCR-ABL mutation and could be a novel drug candidate for treating imatinib-resistant CML patients.

A Novel Role for HMGN1 in Down Syndrome Acute Lymphoblastic Leukemia

Background Down Syndrome (DS) Acute Lymphoblastic Leukemia (ALL) patients have extremely poor outcomes with mortality rates four times greater than non-DS ALL patients within their first two years of diagnosis. They are more suspectible to treatment related toxicities and experience higher relapse rates compared to other ALL patients. Approximately 60% of DS-ALL patients harbor rearrangement of cytokine receptor like factor 2 (CRLF2r), specifically P2RY8-CRLF2, and/or the CRLF2 F232C activating mutation. These lesions are considered poor risk and currently no targeted therapy exist. How increased chromosome 21 gene dosage affect disease phenotype is not yet fully elucidated. However, the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) on chromosome 21, which competes with histone H1 to bind the nucleosome and results in gene activation may be a candidate for targeted therapy in DS-ALL. Methods We aimed to determine the role of HMGN1 in CRLF2r DS-ALL. To model CRLF2r DS-ALL, the trisomy 21 cell line, SET-2, was transduced with a retroviral vector encoding the CRLF2 F232C activating mutation. Gene knockdown of HMGN1 using CRISPR/Cas9 was performed in the SET-2 CRLF2r line and the non-trisomy-21, non-CRLF2 expressing Jurkat line. Individual knockdowns of another two genes on chromosome 21, DYRK1A and ERG were also performed. Knockdown of JAK2 was used as a control as it is critical for CRLF2 signaling. CellTiter-Glo was used to investigate proliferation of knockdown lines to test the hypothesis that HMGN1 is essential for CRLF2r DS-ALL cell proliferation. Lentiviral vectors encoding the P2RY8-CRLF2 fusion gene, CRLF2 F232C activating mutation or an overexpression construct of HMGN1 were transduced into BaF3 cells individually or in combination to test the hypothesis that overexpressing HMGN1 is associated with activation of CRLF2. Quantitative PCR (qRTPCR) for CRLF2 and flow cytometry for the CRLF2/IL7Rα receptor (TSLPR) were used to determine the effect of increased HMGN1 on CRLF2 expression. AnnexinV/7-AAD cell death assays were performed to determine if the effects of HMGN1 could be reduced by the demethylase inhibitor GSK-J4. Results Knockdown of HMGN1 resulted in an 80-90% decrease in HMGN1 protein expression in SET-2 CRLF2 and Jurkat lines compared to the Cas9 controls. While knockdowns of DYRK1A and ERG did not impair the proliferation of SET-2 CRLF2 cells, HMGN1 and JAK2 knockdowns led to a complete proliferation arrest over a period of 120hrs (p=<0.001, n=3), demonstrating their effect on cell division. However, no change in proliferation was observed in the Jurkat knockdown lines. Overexpression of either HMGN1 or P2RY8-CRLF2 alone in BaF3 cells did not result in cytokine independent transformation. However, cytokine independence was triggered in BaF3 cells when HMGN1 and P2RY8-CRLF2 were co-expressed (p=<0.001, n=3); demonstrating a role for HMGN1 in leukemic transformation. Importantly, the overexpression of HMGN1 in the BaF3 P2RY8-CRLF2 line increased the mRNA expression of CRLF2 by 5.8-fold compared to the BaF3 P2RY8-CRLF2 line without HMGN1 (p=0.034, n=3) and increased the mean fluorescence intensity of TSLPR by flow cytometry from 42 to 308 (p=0.008, n=3) (figure 1.a-c) indicating a novel role for HMGN1 in P2RY8-CRLF2 activation. While there are no pharmacological inhibitors for HMGN1, Lane et al. (2014) have shown that the restoration of H3K27 methylation using the demethylase inhibitor GSK-J4 was able to prevent DS-ALL cells from repassaging. Therefore, we have employed the inhibitor GSK-J4 to determine if it can reduce cell survival in HMGN1 overexpressed BaF3 cells. Specific inhibition of BaF3 P2RY8-CRLF2 HMGN1 cells was evident by decreased cell viability at a concentration of 3.8µM compared to BaF3 P2RY8-CRLF2 or BaF3 HMGN1 lines (p=<0.001, n=3) (figure 4.d). Thus, demonstrating a role for HMGN1 in the modification of the P2RY8-CRLF2 methylome and suggesting HMGN1 as a potential therapeutic target. Conclusion These data support the hypotheses that HMGN1 has a significant role in DS-ALL cell proliferation and that overexpression of HMGN1 results in activation of P2RY8-CRLF2. This is the first report of a novel role for HMGN1 in P2RY8-CRLF2 activation and leukemic transformation in CRLF2r DS-ALL. Additionally, we show that HMGN1 is a potential candidate for the development of a pharmacological inhibitor for CRLF2r DS-ALL. Disclosures Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau.

Activation of JAK2/STAT5 Pathway Reduces Expression Level of DNMT3a in MPN Cell Line

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to acute myeloid leukemia (AML), and JAK2V167F is the most frequent MPN driving mutation detected in >95% of PV and 50-60% ET and PMF. DNMT3A is a de novo DNA methyltransferase that catalyzes the addition of methyl groups into active chromatin in CpG-rich regions leading to gene inactivation. Dnmt3a-/- HSC have enhanced self-renewal and a block in differentiation in vivo. Previous study showed that JAK2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation, while whether JAK2V617F regulates DNMT3a still remains unclear. AZ960 is a potent and selective ATP competitive inhibitor of the JAK2 kinase, and previous studies reported that AZ960 possessed the activity selectively against JAK2. LY2784544 has been identified as a selective inhibitor of JAK2V617F and has undergone clinical trials for the treatment of several myeloproliferative disorders. Methods: Empty vector (control) and mutant JAK2V617F were transduced into BaF3 cells using a lentivirus system. JAK2V617F-expressing BaF3 cells grow IL-3 independent and were selected by fluorescence-activated cell sorting (FACS) for GFP expression. The protein expression levels of p-STAT5 and DNMT3a were detected by western blotting. JAK2V617F-expressing and control BaF3 cells were incubated with gradient concentration of LY2784544 or AZ960 to inhibit JAK2/STAT5 pathway. Results: The expression levels of p-STAT5 were obviously up-regulated in the JAK2V617F-expressing BaF3 cells, and DNMT3a was down-regulated. After 1-hour incubation in the serial diluted LY2784544, p-STAT5 were reduced in JAK2V617F-expressing BaF3 cells, with expression of DNMT3a elevated. To further confirm the correlation between JAK2/STAT5 pathway and expression of DNMT3a, another JAK2 inhibitor AZ960 was tested similar to LY2784544. With p-STAT5 expression suppressed, protein level of DNMT3a showed significantly promotion. Conclusion: We observed that JAK2V167F mutation suppresses protein expression levels of DNMT3a in MPN cell lines. JAK2 inhibition by AZ960 and LY2784544 significantly improved expression levels of DNMT3a. The activation of JAK2/STAT5 pathway reduces expression level of DNMT3a in MPN cell line, and the specific mechanism still needs to be explored. Figure Disclosures No relevant conflicts of interest to declare.

Novel CD33 Antibodies Unravel Localization, Biology, and Therapeutic Implications of CD33 Isoforms

Background: Recent promising results from the CD33-directed immunoconjugate, gemtuzumab ozogamicin (GO), has transformed the realm of acute myeloid leukemia (AML) treatment strategies and has opened the door for several other CD33-directed immunotherapeutic strategies to be tested in clinical settings. Starting from accelerated approval in 2000, GO was discontinued in 2010 due to high levels of death in a major phase 3 clinical trial until follow-up efficacy studies allowed for its re-approval in 2017. Despite the complex path of GO as a therapeutic, its impact in the field has been unprecedented and thus ongoing studies seek to improve treatment strategies using GO. Among the most prevalent challenges to this goal is the variation in structure and expression levels of CD33. In particular, previous studies from our group reported rs12459419 C>T, a splicing SNP, resulting in formation of a shorter CD33 isoform (CD33D2) that lacks the critical immunogenic IgV domain (Lamba, 2017). Given that GO recognizes the IgV domain, its therapeutic effect is compromised in patients expressing CD33D2 isoform (CT and TT genotypes). The lack of benefit observed in rs12459419 heterozygous patients is perplexing and warrants in depth investigations to understand biology of CD33D2 isoform. However, the lack of a specific antibody that can recognize the CD33D2 isoform is a significant roadblock to advancing the: (1) understanding of CD33D2 biology; (2) evaluation of CD33D2 isoform as a novel drug target; and (3) explanation regarding why heterozygous patients have compromised response to GO, despite having intermediate levels of CD33FL isoform. Methods: To overcome this roadblock, we focused on developing novel CD33 antibodies specifically targeted to the shared IgC domain between both isoforms (Figure 1). In brief, peptides spanning IgC domain of CD33 were used as immunogens in either Balb/c or C57BL/6 mice for antibody generation. The novel antibodies, named 5C11-2 and HL2541, were used to assess surface-level, intracellular expression, and intracellular localization of CD33 isoforms. The newly developed IgC targeting antibodies and the well-established IgV targeting P67.6 antibody were tested by flow cytometry and western blotting on the CD33 negative BaF3 cell line and BaF3 cells engineered to express CD33FL (BaF3-CD33FLGFP) and CD33D2 (BaF3-CD33D2GFP), as well as AML cell lines representing different rs1245419 genotypes. Results: As expected, naïve Baf3 cells showed no GFP fluorescence signal whereas BaF3-CD33FLGFP, BaF3-CD33D2GFP were positive for GFP confirming expression of CD33 isoforms (Figure 2A). Subsequent staining with P67.6 only produced a positive signal in BaF3-CD33FLGFP cells while BaF3-CD33D2GFP, which lack the IgV domain, were negative (Figure 2B). Interestingly both novel IgC targeting antibodies, 5C11-2 and HL2541, only stained positive for BaF3-CD33D2GFP cells and were negative for BaF3-CD33FL cells (Figure 2C). Western blotting confirmed the recognition of both CD33FL and CD33D2 isoforms by the IgC targeting novel antibody 5C11-2 (Figure 3). These results indicate a potential steric hindrance caused the bulky nature of the clustered extracellular Ig-folds that may have impacted recognition of native CD33FL by IgC targeting antibodies. Cytometry analysis using 5C11-2 on MV4-11 cells, which are heterozygous for the rs12459419 SNP, showed fluorescence signal when stained IgV specific P67.6 but were negative for the IgC specific novel antibody (Figure 4). These results warrant further investigation to establish the exact localization of CD33D2 in context of different cell types and primary AML cells from patients. Nonetheless, our results report successful development of novel CD33 antibodies that recognizes CD33D2 isoform, which holds promise in establishing CD33 biology and understanding the therapeutic implications such as mechanisms contributing to compromised response in rs12459419 heterozygous patients. Disclosures No relevant conflicts of interest to declare.

The Clinical and Functional Relevance of the Transforming AKT E17K Mutation in T-ALL

INTRODUCTION. The importance of IL-7R signaling has been thoroughly studied in the context of both normal T-cell development and leukemogenesis. Upon IL-7 induced heterodimerization of the IL-7Rα and the γc chain, downstream signaling routes regulate survival and proliferation of differentiating T-cells. JAK-STAT, MAPK-ERK and PI3K-AKT pathways are aberrantly activated in T-cell acute lymphoblastic leukemia (T-ALL) patients by recurrent gain-of-function mutations of the IL-7R signaling pathway. IL-7R signaling mutations are a result of mutations in IL7Ra, JAK1/3, PTPN11, NF1, STAT5B, N/KRAS, PI3K or AKT that are found in nearly 35% of pediatric T-ALL patients and are associated with steroid resistance and inferior event-free survival. Here we focus on downstream AKT activation in relation to cellular transformation and steroid resistance. We previously demonstrated that AKTE17K transforms IL3-dependent BaF3 cells in contrast to wild type AKT, while wild type AKT drives steroid resistance in SUP-T1 T-ALL cells in contrast to mutant AKTE17K. AIM. Our aim was to identify functional differences between AKT and AKTE17K molecules in respect to cellular transformation and steroid resistance. We assessed activation level, cellular localization and downstream signaling events that may explain these functional differences. RESULTS. For this study, we used doxycycline-inducible SUP-T1 cell lines that overexpress wild type AKT or mutant AKTE17K molecules. Using immunohistochemistry, we revealed abundant cytoplasmic localization of wild type AKT but predominant plasma membrane localization of AKTE17K and extends previous findings by others. Furthermore, we found evidence for phosphorylation of wild type AKT on serine-473 (S473) and threonine-308 (T308) residues in both cytoplasmic and nuclear fractions, indicating that part of induced AKT becomes activated. In contrast, expression of AKTE17K was accompanied with abundant phosphorylation of T308 while having relatively low levels of S473 phosphorylation. The T308/S473 phosphorylation ratio was 5 times higher for AKTE17K compared to wild type AKT. These results are in line with current views on AKT activation that S473 phosphorylation facilitates the opening of the AKT pleckstrin homology domain and membrane docking, after which AKT is phosphorylated (activated) on the T308 residue by PDK1. The constitutive plasma membrane localization and open conformation of AKTE17K as a result of its E17K pleckstrin homology domain mutation leaves it independent of S473 phosphorylation and results in enhanced T308 phosphorylation by PDK1. Consequently, AKTE17K mutant cells may exhibit reduced cellular sensitivity to allosteric AKT inhibitors but not to type I ATP-competitive inhibitors. Indeed, SUP-T1 cells expressing wild type AKT or mutant AKTE17K are equally sensitivity to the ATP-competitive AZD5363 inhibitor, but AKTE17K cells display reduced sensitivity to the MK2206 allosteric inhibitor compared to AKT wild type cells. In an attempt to explain enhanced steroid resistance by expression of wild type AKT but not mutant AKTE17K, we found no differences in AKT-mediated S134 phosphorylation of the NR3C1 steroid receptor or impaired nuclear import of NR3C1 following steroid treatment (Piovan et al., Cancer Cell 2013). This indicates that AKT induces steroid resistance by other mechanisms in our SUP-T1 cell system. CONCLUSIONS. In contrast to wild type AKT overexpression, we demonstrated that AKTE17K has (i) transforming activity, (ii) does not yield cellular resistance to steroid treatment, (iii) is predominantly localized at the plasma membrane, (iv) has high T308 over S473 phosphorylation and (v) is less sensitive to the MK2206 allosteric AKT-inhibitor. These observed differences are likely caused by the constitutively open AKTE17K conformation. Since no differences in NR3C1 phosphorylation or nuclear shuttling were observed in our model, we hypothesize other mechanisms underlie the provoked steroid resistance by AKT wild type overexpression. To identify these novel mechanisms, we are currently performing an unbiased phospho-protein array to identify differentially phosphorylated AKT substrates. Disclosures No relevant conflicts of interest to declare.

PRMT5 Inhibition Modulates E2F1 Methylation and Gene Regulatory Networks Leading to Therapeutic Efficacy in JAK2VF Mutant MPN

Dysregulation of the protein arginine methyltransferase, PRMT5, occurs in a variety of malignancies and is associated with increased cancer cell proliferation. We investigated the role of PRMT5 in MPN pathogenesis through use of C220, a potent and selective small molecule PRMT5 inhibitor, and aimed to elucidate key PRMT5 targets contributing to the maintenance of MPN cells. We found that PRMT5 is overexpressed in MPN, most highly in PV. C220 dose-dependently reduced the proliferation of MPN cell lines and BaF3 cells expressing JAK2V617F in vitro. Human CD34+ cells from PV/PMF patients showed a high degree of sensitivity to C220 ex vivo. Consistent with the in vitrostudies, C220 treatment of SET2 xenografts in vivoled to a 71% reduction of median tumor volume, greater than that observed with JAK inhibition (59%). We assessed the efficacy of C220 in Jak2V617F- and MPLW515L-driven murine MPN models.In the conditional Jak2V617Fmodel of PV, mice treated with C220 displayed significant lower hematocrits (median 50.1% vs. 77.4 %, p<0.0001), WBC (median 4.5 K/µl vs. 16.9 K/µl, p<0.0001), reticulocytes (median 73 K/µl vs. 1522 K/µl, p<0.0001) and splenomegaly (median weight 68 mg vs. 270 mg, p<0.0001) compared to vehicle treatment. CD105+CD150- progenitors, committed erythroid progenitors (Pre-CFU-E), MEP, CD71-Ter119+ pro-erythroblasts and CD71+/Ter119+ erythroblasts were significantly decreased with C220 treatment [15.7% (p<0.0001), 36% (p=0.0004), 58% (p=0.01), 10% (p<0.0001) and 11% (p<0.0001), respectively, relative to vehicle]. In the MPLW515Ladoptive transfer model of ET/MF, C220 attenuated leukocytosis (WBC 11.1 K/µl vs. 155.3 K/µl, p=0.023), splenomegaly (136 mg vs. 423 mg, p=0.003), hepatomegaly (1118 mg vs. 1677 mg, p=0.004), and BM fibrosis (reticulin score 0.5 vs. 2+). C220 significantly reduced serum cytokines in the Jak2V617F(Eotaxin, GM-CSF, IL-12p40, LIX, KC, MCP1, MIP1a, MIP-2, M-CSF, RANTES, IP-10, IL-1b) and MPLW515L(MIP1b, TNF- alpha, RANTES, IP-10 and IL-1b) model consistent with inhibition of systemic inflammation characteristic of MPN. We next assessed the efficacy of combined PRMT5 and JAK inhibition. Dual targeting of JAK and PRMT5 displayed additive effects on SET2 cell proliferation in vitro and was superior to monotherapy with either agent in vivo. In the Jak2V617Fmodel, dual JAK/PRMT5 inhibition further reduced key features such as splenomegaly (median weight 39 mg vs. 68 mg or 127 mg, p=0.0004 and p<0.0001), elevated hematocrit (median 41.1% vs. 50.1% or 85.1%, p=0.0089 and p=0.0009), WBC (median 2.1 K/µl vs. 4.5 K/µl or 7.4 K/µl, p=0.0015 and p<0.0001) and led to a significant decrease in erythroid progenitor populations compared to PRMT5 or JAK inhibitor monotherapy. Dual JAK/PRMT5 inhibition showed increased efficacy in the MPLW515L model with more significant suppression of leukocytosis (median WBC 3.1 K/µl vs. 176.7 K/µl, p=0.0436), splenomegaly (median weight 117 mg vs. 438 mg, p=0.0062) and hepatomegaly (median weight 1105 mg vs. 1573 mg, p=0.0188). To further understand the mechanism by which C220 leads to therapeutic efficacy in MPN, we performed RNA-seq in sorted MEP from the Jak2V617FBMT model and from SET2 xenografts after vehicle or C220 in vivotherapy. GSEA revealed E2F targets as one of the major gene sets down regulated upon treatment with C220. Consistent with the potent suppression of E2F1 target gene expression in vivo, we found that E2F1 and PRMT5 physically interact in JAK2-mutant MPN cells. Moreover, PRMT5 inhibition altered the methylation status of E2F1, leading to reduced expression of E2F1 downstream targets, including genes involved in cell cycle and DNA damage repair. Functional investigation of key downstream targets is ongoing. Our data provide evidence that PRMT5 is a credentialed therapeutic target in MPN, alone and in combination with JAK inhibition. Moreover, the therapeutic effects of PRMT5 inhibition in MPN include attenuated expression of E2F targets, which our data suggests is mediated by PRMT5 methylation of E2F1. These data demonstrate a novel link between PRMT5, E2F1 gene regulatory function, and the survival of MPN cells and provide a strong mechanism-based rationale for therapeutic studies of PRMT5 inhibitors in MPN. Based on these studies and others, PRT543, a novel and selective PRMT5 inhibitor, is currently being evaluated in a Phase I clinical trial in advanced cancers, including myelofibrosis (NCT03886831). Disclosures Bhagwat: Prelude Therapeutics: Employment. Viny:Hematology News: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Other: Sponsored travel. Grego:Prelude Therapeutics: Employment. Mehta:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Levine:Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Celgene: Consultancy, Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Lilly: Honoraria; Amgen: Honoraria.

Germline Mutations Predispose to Familial Myeloproliferative Neoplasms

Background: Myeloproliferative neoplasms (MPNs), commonly polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET), are a group of malignant hematologic disorders characterized by proliferation and accumulation of terminally differentiated blood cells of erythroid, myeloid and megakaryocytic origin (Cazzola, Blood 2014).Somatic driver mutations, frequently JAK2V617F, MPLW515L/K or mutant CALR in sporadic MPNs affect signaling by the thrombopoietin (THPO) and/or the erythropoietin (EPO) receptor (Pickman, PLoS 2016). MPL is the receptor of THPO and JAK2 is the key downstream mediator of THPO/MPL signaling (Nangalia NEJM 2013). In familial MPNs (families with more than 1 member developing a MPN) a germline mutation predisposes to disease development. These mutations can reveal pathways or processes involved in the pathogenesis of the disease, independent of, or cooperating with, the somatic driver mutations (Rumi, JCO 2007). We identified 3 putative predisposing germline mutations in 1 family, 3 affected generations developed MPN (PMF, ET and PV): RBSN frameshift,JAK2R340Q & PRKAA2Q425P missense variants. RBSN encodes Rabenosyn-5 protein (RBSN) which plays a role in early growth-factor receptor clathrin-dependent endocytosis (Nielsen, J Cell Biol 2000) (Fig.1).We discovered that wild type (WT) Rabenosyn-5 regulates cell surface MPL levels in hematopoietic progenitor cells and predict that MPL levels on the cell surface are negatively associated with WT Rabenosyn-5 levels. We hypothesize that the RBSN mutation predisposes to MPN development in this family, leading to abnormal activation of THPO signaling by reducing MPL turnover. Methods: Peripheral blood DNA from patient samples were analyzed for potential putative mutations using Agilent SureSelect Human ALL Exon V5+UTRs exome capture kit followed by parallel sequencing with Illumina HiSeq 2000. In vitro studies involved using the BaF3 system and CRISPR-Cas9 gene editing technique. We generated BaF3-MPL (BaF3 cells transduced with human CD4-MPL fusion) cell lines that rely on IL3 or human THPO cytokines for growth & proliferation (Fig.2&3). We transduced RBSN into the BaF3-MPL cell lines and produced RBSN heterozygous (HT) and homozygous (HM) mutants. We first generated LentiCRISPRV2GFP-mRBSN vector by inserting RBSN specific guide RNA into the LentiCRISPRV2GFP vector and generated lentivirus expressing both Cas9 and guide RNA by co-transfecting 293T cells with LentiCRISPRV2GFP vector and viral packaging vectors. The final virus was used to infect BaF3-MPL cells. Transduced cells were purified by FACS to select GFP+ cells and individual cells were grown in culture. RBSN gene mutations were examined using PCR and sequenced accordingly. To study whether the RBSN mutation alters the surface MPL levels, we stained BaF3-MPL, HT-RBSN-BaF3 and HM-RBSN-BaF3 cells with APC-CD4 antibody and the surface levels of MPL were detected by FACS by comparing the mean fluorescence intensity (MFI) of APC. Results: We identified several colonies with either HT-RBSN-BaF3 or HM-RBSN-BaF3. The guide RNA we designed specifically targeted the 12th exon of the RBSN gene which best recapitulates the mutations observed in our patients, thus, the frameshift mutations in HT-RBSN-BaF3 and HM-RBSN-BaF3 cell lines we predict will make truncated forms of RBSN which will be verified by Western Blotting for future experiments. Surface levels of MPL in the HT-RBSN-BaF3 and HM-RBSN-BaF3 cells are greater than that in BaF3-MPL cells, suggesting RBSN negatively regulates THPO-MPL signaling by regulating the cell surface MPL levels (Fig. 4). Conclusions: RBSN is a regulator of receptor trafficking, which mediates the fusion of the endocytosed receptor to the early endosome and then lysosome for degradation. We propose that genetic inactivation of RBSN might prevent the endocytosed MPL from endosome-degradation and enhance the reuse of MPL by recycling. Future experiments are planned to include in vivo studies to study whether inactivation of RBSN promoted MPN-like disease development into NSG mice and transducing our established cell lines to label individual cell compartments to study THPO-stimulated signaling. Our findings, if confirmed, will further clarify aspects of familial and somatic MPN pathogenesis and may inform efforts to devise new therapeutic and diagnostic strategies for this disease complex. Disclosures No relevant conflicts of interest to declare.

RUNX2 Regulates Cell Migration in T-Cell Lineage Acute Lymphoblastic Leukemia

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy accounting for 10-15% of pediatric ALLs. While cure rates have recently improved, event free survival is <10% in patients with relapsed disease. We recently showed that Runt related transcription factor 2 (RUNX2) is upregulated in high-risk T-ALL including patients harboring KMT2A-R (p< 0.0001), ETP phenotype (p< 0.0001) and those who failed induction therapy (p <0.005) (Sharma et al., AACR Annual Meeting 2018; #4129). We also demonstrated that RUNX2 positively regulates T-ALL survival via Akt/β-catenin/survivin axis and observed a significant increase in survival of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice engrafted with shRNA-RUNX2 depleted T-ALL cells (median survival 52 days) compared to animals engrafted with negative control cells (median survival 38 days; p< 0.0001). Because RUNX2 is involved in invasiveness and metastasis of many solid tumors, we hypothesized that RUNX2 might modulate the migration and dissemination of T-ALL. To test the regulatory effects of RUNX2 on T-ALL migration we used lentiviral transduction to induce or knock down RUNX2 expression in T-ALL cell lines (n = 4) and/or primary patient samples (n = 3), and/or BaF3 cells expressing KMT2A-R (KMT2A-MLLT4 or KMT2A-MLLT1). While induced expression of RUNX2 had no effect on T-ALL cell growth and cell cycle, we discovered that RUNX2 positively regulated the expression of CXCR4 receptor and migration of T-ALL cells towards CXCL12 chemokine (p< 0.0005). Because cell migration is overall an energy-demanding process, we used the Agilent Seahorse XF Analyzer to study leukemic cell metabolism. We found that RUNX2 upregulation leads to increased oxygen consumption rate and ATP production (p< 0.0001) as well as the increase in basal and compensatory glycolysis (p< 0.005), suggesting that RUNX2 potentiates metabolic activity of T-ALL cells. We next utilized flow-based Mito-Tracker Red CMXRos and Reactive Oxygen Species (ROS) assays to examine the impact of RUNX2 on mitochondrial activity. Forced expression of RUNX2 increased mitochondrial membrane potential corresponding to decreased ROS levels. RUNX2 positively regulated the expression of critical regulators of mitochondrial fission (P-DRP1 Ser616, DRP1, MFF and FIS1), fusion (OPA1 and Mfn1, Mfn2) and mitochondrial biogenesis (PGC1α and NRF1). In converse experiments, RUNX2 depletion reduced the oxidative phosphorylation, glycolytic flux and mitochondrial activities in the tested T-ALL cells suggesting that RUNX2 displays unique metabolic signatures that might enhance leukemia aggressiveness. Disruption of mitochondrial fission with Midivi-1 inhibitor (1 μΜ, 16 hr) reduced RUNX2-mediated T-ALL migration towards CXCL12 (P< 0.0001) suggesting that RUNX2 regulates mitochondrial dynamics to fosters T-ALL cell migration in vitro. Similarly, treatment with 2-deoxy-D-glucose (2DG), a glucose analog reduced the levels of migrating T-ALL cells (p< 0.005) indicating the roles of metabolic reprograming in RUNX2-mediated T-ALL cell migration. Our results suggest that RUNX2 regulates mitochondrial dynamics and cell metabolism to promote T-ALL migration. We propose deregulation of RUNX2 expression as a novel mechanism governing T-ALL migration and dissemination. Ongoing in vivo studies will reveal the roles of RUNX2 in T-ALL infiltration to extramedullary tissue. Disclosures Loh: Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Hunger:Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Jazz: Honoraria; Novartis: Consultancy.

VEGF-A-Cleavage by FSAP and Inhibition of Neo-Vascularization.

Alternative splicing leads to the secretion of multiple forms of vascular endothelial growth factor-A (VEGF-A) that differ in their activity profiles with respect to neovascularization. FSAP (factor VII activating protease) is the zymogen form of a plasma protease that is activated (FSAPa) upon tissue injury via the release of histones. The purpose of the study was to determine if FSAPa regulates VEGF-A activity in vitro and in vivo. FSAP bound to VEGF165, but not VEGF121, and VEGF165 was cleaved in its neuropilin/proteoglycan binding domain. VEGF165 cleavage did not alter its binding to VEGF receptors but diminished its binding to neuropilin. The stimulatory effects of VEGF165 on endothelial cell proliferation, migration, and signal transduction were not altered by FSAP. Similarly, proliferation of VEGF receptor-expressing BAF3 cells, in response to VEGF165, was not modulated by FSAP. In the mouse matrigel model of angiogenesis, FSAP decreased the ability of VEGF165, basic fibroblast growth factor (bFGF), and their combination, to induce neovascularization. Lack of endogenous FSAP in mice did not influence neovascularization. Thus, FSAP inhibited VEGF165-mediated angiogenesis in the matrigel model in vivo, where VEGF’s interaction with the matrix and its diffusion are important.

Discovery of a highly selective VEGFR2 kinase inhibitor CHMFL-VEGFR2-002 as a novel anti-angiogenesis agent

Angiogenesis is an essential process in tumor growth, invasion and metastasis. VEGF receptor 2 (VEGFR2) inhibitors targeting tumor angiogenic pathway have been widely used in the clinical cancer treatment. However, most of currently used VEGFR2 kinase inhibitors are multi-target inhibitors which might result in target-associated side effects and therefore limited clinical toleration. Highly selective VEGFR inhibitors are still highly demanded from both basic research and clinical application point of view. Here we report the discovery and characterization of a novel VEGFR2 inhibitor (CHMFL-VEGFR2-002), which exhibited high selectivity among structurally closed kinases including PDGFRs, FGFRs, CSF1R, etc. CHMFL-VEGFR2-002 displayed potent inhibitory activity against VEGFR2 kinase in the biochemical assay (IC50 = 66 nmol/L) and VEGFR2 autophosphorylation in cells (EC50s ∼100 nmol/L) as well as potent anti-proliferation effect against VEGFR2 transformed BaF3 cells (GI50 = 150 nmol/L). In addition, CHMFL-VEGFR2-002 also displayed good anti-angiogenesis efficacy in vitro and exhibited good in vivo PK (pharmacokinetics) profile with bioavailability over 49% and anti-angiogenesis efficacy in both zebrafish and mouse models without apparent toxicity. These results suggest that CHMFL-VEGFR2-002 might be a useful research tool for dissecting new functions of VEGFR2 kinase as well as a potential anti-angiogenetic agent for the cancer therapy.

Targeting glutaminase1 and synergizing with clinical drugs achieved more promising antitumor activity on multiple myeloma.

Multiple myeloma (MM) pathogenesis remains incompletely understood and biomarkers predicting treatment response still remain lacking. Here we describe the rational mechanisms of combining targeting glautaminase1 (GLS1) with other chemo-reagents for MM treatment. Gls1 is highly expressed cMYC/KRAS12V-drived plasmacytoma (PCT) cells. Down-regulation of Gls1 with miRNAi in cMYC/KRAS12V-expressing BaF3 cells prevented them from growing independence of interleukin 3 (IL3). By using our cMYC/KRAS12V-transduced adoptive plasmacytoma mouse model, we found that Gls1 is involved in PCT pathogenesis. Down-regulation of Gls1 significantly prolonged the survival of PCT recipients. Knockdown of Gls1 increased the expression of Cdkn1a and Cdkn1b and decreased the expression of some critical oncogenes for cancer cell survival, such as c-Myc, Cdk4, and NfκB, as well as some genes which are essential for MM cell survival, such as Irf4, Prdm1, Csnk1α1, and Rassf5. Combination of Gls1 inhibition with LBH589, Bortezomib, or Lenalidomide significantly impaired tumor growth in a MM xenograft mouse model. Our data strongly suggest that Gls1 plays an important role for MM pathogenesis and that combination of GLS1 inhibitor with other MM therapy agents could benefit to MM patients.

2-Oxo-3,4-dihydropyrimido[4,5-d] pyrimidines as new reversible inhibitors of EGFR C797S (Cys797 to Ser797) mutant

Extensive structure-activity relationships (SARs) study of JND3229 was conducted to yield a series of new reversible 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidine privileged scaffold as EGFRC797S inhibitors. One of the most potent compound 6i potently suppressed EGFRL858R/T790M/C797S kinase with an IC50 value of 3.1nmol/L, and inhibited the proliferation of BaF3 cells harboring EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S mutants with IC50 values of 290nmol/L and 316nmol/L, respectively. Further, 6i dose-dependently induced suppression of the phosphorylation of EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S in BaF3 cells. Compound 6i may serve as a promising lead compound for further drug discovery overcoming the acquired resistance of non-small cell lung cancer (NSCLC) patients.