Kooperativer Bibliotheksverbund

In: International Journal of Reproduction, Contraception, Obstetrics and Gynecology, Medip Academy, Vol. 9, No. 6 ( 2020-05-27), p. 2393-

Abstract: Background: Viral hepatitis is the most common liver disease in pregnancy and is also the most common cause of jaundice in pregnancy in tropical countries. Risk factors for transmission are intravenous drug abuse, surgical and dental procedures done without adequate sterilization of instruments, sexual route etc. Early diagnosis and management can prevent maternal and fetal complications. This study was done to evaluate the frequency, risk factors and pregnancy outcome in hepatitis B virus (HBV) and hepatitis C virus (HCV) positive antenatal women.Methods: This case control study was conducted in Teerthankar Mahaveer Medical College and Research Centre, Moradabad, Uttar Pradesh, India from January 2017 to June 2018 on total 2511 pregnant women. The serum samples were checked for presence of hepatitis B surface antigen (HBsAg) and presence of IgG antibodies to HCV. Analysis of sociodemographic profile, risk factors and pregnancy outcome were done in all HBV and HCV positive women.Results: Out of 2511 pregnant women, 292 were tested positive for hepatitis. Maximum number of women were in the age group of 21-30 years. Most of the seropositive women were multipara. Frequency of positivity was maximum for HCV (67.1%). The risk factors for transmission in study population were intravenous drug abuse, blood transfusion, history of surgery and tattooing.Conclusions: Hepatitis infection rate is increasing. Universal screening for HBV and HCV can be recommended in pregnant women in developing countries. Education and awareness of public and health care workers can reduce the risk of transmission. 

Type of Medium: Online Resource

ISSN: 2320-1789 , 2320-1770

URL: Article

DOI: 10.18203/2320-1770.ijrcog20202317

Language: Unknown

Publisher: Medip Academy

Publication Date: 2020

In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 1006-1006

Abstract: Background: Resistance to imatinib in patients with chronic myeloid leukemia (CML) is commonly associated with reactivation of BCR-ABL due to kinase domain mutations or increased expression. Activation of SRC family kinases has been shown in isolated patients without evidence of BCR-ABL reactivation. We hypothesized that activating mutations of the RAS pathway (one of the major downstream mediators of BCR-ABL) or of leukemia-associated tyrosine kinases other than BCR-ABL may cause BCR-ABL-independent resistance. Patients and Methods: CML patients with imatinib resistance were initially screened for BCR-ABL mutations. Two sets of nested primers were used to specifically amplify BCR-ABL. Sequence analysis covered the entire 5′ end of ABL including the Cap, SH3, SH2 and kinase domains. Bone marrow samples from patients without BCR-ABL mutations (N=17) were screened for mutations of NRAS, KRAS and FLT3 by direct DNA sequencing. Analysis of PTPN11 and PDGFR is ongoing and will be presented. Results: We detected a KRAS exon 3 mutation (T58I) in 1/17 patients (5.8%) and confirmed this mutation by 2-directional sequencing of 3 independently amplified PCR products. No mutations in NRAS or FLT3 were observed. Analysis of archived samples revealed that T58I was absent while the patient was responding to 800mg of imatinib and achieved a major cytogenetic response (MCyR), became detectable before relapse and remained detectable (at approximately 30%) in 4 subsequent samples collected over a period of 18 months (Table 1), while the patient achieved a second MCyR on dasatinib. The T58I allele became undetectable prior to relapse on dasatinib. None of the samples tested were positive for BCR-ABL kinase mutations. T58I is an activating mutation of KRAS that has been detected in patients with Noonan syndrome who developed JMML (Schubbert et. al, Nature Genetics, 2006, 38:331). We tested the functional relevance of T58I in the context of imatinib resistance in 32D cells co-expressing T58I and BCR-ABL, with KRAS G12D and native KRAS as controls. Sensitivity to imatinib was reduced (G12D 〉 T58I 〉 native), with 15% 32D cells expressing KRAS T58I retaining viability at 2.5 μM over 3 Days. These data demonstrate that T58I confers partial imatinib resistance, although it does not induce complete growth factor independence. Conclusion: Activating KRAS mutations may contribute to resistance or disease persistence in CML patients treated ABL kinase inhibitors. Table 1: Follow-up summary of patient with KRAS T58I mutation Sample date Treatment prior to cytogenetic evaluation (mg/day) Cytogenetics (Ph+ metaphases) FISH (Bcr-Abl)% KRAS T58I Allele% Bcr-Abl kinase domain Feb-02 Imatinib 400 11/17 41.5 0 WT Dec-02 Imatinib 400 20/20 78 0 WT Dec-03 Imatinib 600–800 +Arsenic trioxide 5/25 31.5 0 WT Oct-04 Imatinib 800+Arsenic trioxide 7/20 14.5 31.8 WT Jun-05 Imatinib 800 17/20 62.5 ND WT Oct-05 Dasatinib 140 ND 23 28.8 WT Dec-05 Dasatinib 100 6/20 24.5 ND WT Mar-06 Dasatinib 80 1/1 13.5 37.4 WT Apr-06 Dasatinib 80 1/12 10 ND WT Sep-06 Dasatinib 80 3/20 24 34.9 WT Dec-06 Dasatinib 80 1/3 29 33.1 WT Feb-07 Dasatinib 80 4/20 51 0 WT May-07 Dasatinib 80 19/20 60.5 0 WT

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V110.11.1006.1006

Language: English

Publisher: American Society of Hematology

Publication Date: 2007

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 2511-2511

Abstract: The SET oncoprotein, an inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia cells, preventing PP2A from performing its regulatory role in deactivating signaling proteins by dephosphorylation. Restoration of PP2A activity in both chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells to normal levels through shRNA-mediated knockdown of SET results in reduced leukemogenesis. Given the central role of PP2A and SET in regulating various kinase-dependent and -independent downstream signaling pathways, we evaluated the efficacy of SET antagonism in CML and AML cell lines as well as primary patient cells using OP449, a novel, specific, cell-penetrating SET antagonist. Results Treatment of human and murine CML cells with OP449 resulted in dose-dependent increase in PP2A activity and selective inhibition of cell growth (IC50: 0.60 to 1.11 μM), while parental Ba/F3 cells exhibited no measurable cytotoxicity. OP449-mediated decrease in the viability of leukemia cells was significantly rescued by co-treatment with okadaic acid, a PP2A inhibitor, confirming efficacy is mediated through PP2A activation. OP449 was also 3 to 8-fold more potent than FTY720 (a known activator of PP2A) and induced dephosphorylation/degradation of BCR-ABL1, AKT, and STAT5. Importantly, OP449 demonstrated activity against the ABL1 tyrosine kinase inhibitor-resistant BCR-ABL1T315I mutant and the BCR-ABL1E255V/T315I compound mutant (IC50: 1.62 and 1.97 μM, respectively). Consistent with cell line findings, OP449 also inhibited growth of primary cells from CML blastic phase patients harboring either wildtype BCR-ABL1 or BCR-ABL1T315I while normal CD34+ cells exhibited minimal effect. Further, treatment of CML cell lines and primary CD34+ CML cells with OP449 in combination with the ABL1 tyrosine kinase inhibitors showed significantly increased cytotoxicity as compared to each compound alone. For example, treatment of primary CD34+ CML cells with 2.5 μM OP449 or 200 nM nilotinib alone each resulted in a 50% reduction in colony formation, while combination of OP449 and nilotinib at these concentrations reduced colony formation by approximately 87%, suggesting synergistic reduction of clonogenicity (combination index: 0.195). Similar to our findings in CML cells, OP449 increased PP2A activity and suppressed growth in a dose-dependent manner in AML cell lines and primary patient samples harboring various different genetic lesions including FLT3-ITD, CSF1R overexpression, NRASQ61L, and JAK3A572V. Additionally, synergistic inhibition of these cells was observed when OP449 was combined with relevant tyrosine kinase inhibitors and chemotherapy. For example, treatment of MOLM-14 cells (FLT3-ITD) with 2.5 μM OP449 or 1 nM AC220 alone reduced cell viability by 58% and 75%, respectively; combined treatment reduced cell growth 96% (combination index: 0.723). Similarly, treatment of HL-60 cells (NRASQ61L) with 1 μM OP449 or 250 nM cytarabine alone reduced cell viability by 40% and 60%, respectively, whereas combined treatment led to a 94% reduction in viability (combination index: 0.630). Mechanistically, AML patient samples showed significantly increased SET expression compared to normal CD34+ cells, and treatment of AML cells with OP449 reduced phosphorylation of downstream ERK, STAT5, AKT and S6 ribosomal protein signaling. Finally, to evaluate OP449 antitumor efficacy in vivo, we tested OP449 (5 mg/kg intraperitoneally every 3 days) in xenograft mice bearing human HL-60 cell derived tumors. OP449 significantly inhibited tumor growth measured over time and resulted in a 〉 2-fold reduction in tumor burden at the end of the experiment compared to vehicle-treated controls (Day 18: 1.14±0.06 g vs. 0.45±0.08 g, respectively; p 〈 0.001). These results demonstrate the in vivo efficacy of OP449 in a murine leukemia model. Conclusions SET antagonism is selectively cytotoxic to CML and AML cells harboring various genetic lesions and drug-resistant mutations. Our results demonstrate that combined targeting of SET and tyrosine kinases provides more efficient and selective inhibition of leukemia cell growth for a broad range of oncogenic lesions as compared to normal cells. Taken together, our findings suggest a novel therapeutic paradigm of SET antagonism in combination with tyrosine kinase inhibitors for the treatment of CML and AML patients with drug resistance. Disclosures: Agarwal: Oncotide Pharmaceuticals: Research Funding. Tyner:Incyte Corporation: Research Funding. Vitek:Oncotide Pharmaceuticals: Employment. Christensen:Oncotide Pharmaceuticals: Employment. Druker:Ambit Biosciences: Consultancy, PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb/Novartis: Currently PI or co-I on Novartis & Bristol-Myers Squibb clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not derive salary, nor does his lab Other; Oncotide Pharmaceuticals: Research Funding, Subaward from NIH STTR, Subaward from NIH STTR Other.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.2511.2511

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 2603-2603

Abstract: Background: p38 mitogen-activated protein kinase (p38MAPK) is activated by various pro-inflammatory and stress-related stimuli, and has been an attractive therapeutic target for autoimmune diseases. p38MAPK (hereafter referred to as p38) signaling is also involved in cell proliferation, differentiation, apoptosis, and invasion, suggesting that it may be a potential therapeutic target for cancer. We found that inflammatory cytokines, including interleukin-1 (IL-1), promote growth and survival of more than half of the acute myeloid leukemia (AML) patient samples we tested. Since p38 is a downstream mediator of inflammatory pathways, we hypothesized that targeting p38 might be an effective therapeutic strategy in AML and other hematologic malignancies. To test this hypothesis, we evaluated the effectiveness of three p38 inhibitors using in vitro studies in primary AML patient samples. We found that targeting p38 blocks IL-1-activated extrinsic signaling and is a critical therapeutic target in a large subset of AML patients. Methods: We screened ~1000 primary leukemia patient samples for sensitivity to p38 inhibition using varying concentrations of doramapimod (BIRB 796) in a cell growth assay. We compared the sensitivity profile of doramapimod with 2 other small-molecule p38 inhibitors currently in clinical trials: ARRY 614, a dual p38/Tie2 inhibitor, and ralimetinib, which blocks activation of p38 by its substrate MK2. We determined cell viability, survival, and downstream signaling in the presence of 10 ng/ml IL-1α or IL-1β. Patient samples with IC50 〈 1000nM were considered drug responsive. Results: In our patient population, we observed response rates of 31% in AML (109/350), 27% in myelodysplastic syndromes (MDS; 25/93), 19% in myeloproliferative neoplasms (23/123), 13% in mature B-cell neoplasms (30/232), and 10% in precursor lymphoid neoplasms (19/182). Focusing on AML, we compared the sensitivity profile of doramapimod with two other small-molecule p38 inhibitors, ralimetinib and ARRY 614. These inhibitors showed strikingly similar sensitivity profiles to doramapimod when tested in an additional 25 primary AML samples, with ~25% responsive and median IC50 of 11 nM for ARRY 614 (range: 7-650nM), 105 nM for ralimetinib (range: 7-850nM), and 18 nM for BIRB 796 (range: 13-40nM). Because IL-1 is known to stimulate p38 signaling, we compared the response rates for these three p38 inhibitors with or without IL-1 in a dose-response study. IL-1 increased the percent of AML samples responding to p38 inhibition from 25% to 60%, indicating a potentially important role of extrinsic inflammatory stimuli in p38 inhibitor sensitivity. Consistent with this all three p38 inhibitors were similarly effective in blocking the growth of primary AML CD34+ progenitors, suggesting that targeting p38 might reduce early progenitor AML cells. Further, we compared doramapimod, ralimetinib, and ARRY 614 for their ability to inhibit p38 phosphorylation in primary AML samples using flow cytometry and immunoblot analysis; all three inhibitors blocked p38 pathway activation in AML cells. Notably, in clinical studies of ARRY 614 in MDS patients, preliminary biomarker analyses demonstrated persistent inhibition of phospho-p38 in the bone marrow during the treatment. Also, consistent with functional inhibition of p38, there was a profound decrease in plasma cytokine concentrations, most significantly IL-1, during ARRY 614 treatment. In 250 primary AML samples, we observed no correlation between BIRB 796 sensitivity in vitro and clinical metrics such as white blood cell count, blast percentage in peripheral blood or bone marrow, karyotype, or tumor genotype. This suggests that IL-1 and p38 activation might be independent biomarkers of drug sensitivity. Conclusions: These data underscore the importance of the p38MAPK pathway in the pathobiology of AML and provide strong preclinical evidence to support p38MAPK as a therapeutic target. Targeting p38MAPK might also block tumor-extrinsic signaling, as indicated by IL-1-activated signaling. That all three p38MAPK inhibitors showed comparable sensitivity profiles holds promise for ARRY614, which showed the lowest median IC50 and is currently in clinical development. In addition, with further study these findings may be extended to hematologic malignancies other than AML. Disclosures Winski: Array BioPharma Inc.: Employment. Cable:Array BioPharma Inc.: Employment. Tyner:Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding. Agarwal:CTI BioPharma: Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V126.23.2603.2603

Language: English

Publisher: American Society of Hematology

Publication Date: 2015

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 570-570

Abstract: Background: A momentous challenge in the treatment of acute myeloid leukemia (AML) is the significant molecular heterogeneity in driving abnormalities and rapid emergence of resistance. Successful clinical translation of novel targeted agents has been impeded by an incomplete understanding of the genetic drivers and the role of the bone marrow microenvironment that modulates response to therapy. There is thus a need for further investigation of the signaling mechanisms contributing to disease pathogenesis. We previously identified growth dependency on interleukin-1 (IL-1) signaling in 〉 50% of the 90 primary AML samples tested. IL-1 signals through IL-1 receptor-associated kinase (IRAK1), a major mediator of innate immunity and inflammatory responses and a potentially critical therapeutic target in hematopoietic malignancies. These results suggest that targeting the commonly dysregulated IL-1 signaling pathway would be therapeutically beneficial to AML patients. However, there is no clinically viable molecule available to selectively inhibit IL-1 signaling. Here we uncovered that pacritinib, a dual FLT3/JAK2 inhibitor, has high specificity and sensitivity to target IRAK1 in various hematopoietic malignancies, including AML. Methods: We used a combination of biochemical, structural biology, and functional approaches to elucidate the mechanism of action of pacritinib and establish its sensitivity and specificity for target inhibition. Kinome screening analysis against 429 recombinant kinases in the presence of 100 nM pacritinib (approximately 50% of the steady state concentration of free pacritinib at the phase 3 dose of 400 mg QD) was followed by titration (1-100 nM) against those kinases that were 〉 50% inhibited at 100 nM. Because pacritinib is an established dual FLT3/JAK2 inhibitor, we then compared the efficacy of pacritinib to the FLT3 inhibitor quizartinib and the JAK2 inhibitor ruxolitinib in 16 AML cell lines and 25 primary AML samples harboring various genetic lesions. The effects of drug treatments were evaluated on cell viability, survival and downstream signaling. Patient samples having IC50below 1000 nM were considered responsive. To validate the selectivity of pacritinib for binding IRAK1, we identified key interacting residues by molecular docking simulation and performed targeted mutagenesis studies. Results: Pacritinib inhibited the activity of FLT3 and all JAK family members except JAK1 at IC50 values of 〈 50 nM. Interestingly, Pacritinib also inhibited IRAK1 (IC50 =13.6 nM). Pacritinib was comparable to quizartinib and ruxolitinib in inhibiting the growth of FLT3- and JAK2/3-dependent cells, respectively, but also reduced the viability and survival of AML cell lines and primary samples harboring a wide variety of genetic abnormalities, including RUNX translocation and RAS mutations (median IC50 = 300 nM; range = 70-600 nM). We observed a similar trend of pacritinib sensitivity in primary AML samples (median IC50= 130 nm; range = 2.0-1000 nM), with a higher response rate (18/23, or 78%) than that to ruxolitinib (26%) or quizartinib (56%). Interestingly, treatment of primary AML samples with IL-1 sensitized primary AML cells to pacritinib showing a 91% response. The IL-1 dependent growth inhibition was more evident with pacritinib than with either ruxolitinib or quizartinib, suggesting that pacritinib specifically targets this inflammatory pathway. Pacritinib-treated AML cells revealed inhibition of the IL-1 pathway, including IRAK1 and p38MAPK. Consistent with the inhibitory effects of pacritinib on FLT3 and JAK2, we observed inhibition of FLT3 and ERK1/2, and of JAK2/JAK3/STAT5 in FLT3- or JAK-dependent AML cells, respectively. Computational modeling indicated that pacritinib binds to the ATP binding-pocket of IRAK1 making a critical interaction with S295. Homology alignments of IRAK1 with JAK2, FLT3, and CDK2 guided the creation of S295D, DG294, and D298K mutations in IRAK1. These mutations, which cause IRAK1 to resemble the pacritinib-insensitive CDK2, abrogated the inhibitory effect of pacritinib. Conclusions: Pacritinib is a specific inhibitor of IRAK1 that blocks IL-1 signaling in AML-derived cell lines and primary patient samples. To our knowledge, pacritinib is the first clinically viable molecule to robustly and selectively inhibit IRAK1. These findings may be extended to a variety of cancer types with a dependence on IRAK1 kinase. Disclosures Druker: Novartis Pharmaceuticals: Research Funding; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; ARIAD: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; Oregon Health & Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; McGraw Hill: Patents & Royalties; Millipore: Patents & Royalties; Sage Bionetworks: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Myers Squibb: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Singer:CTI BioPharma, Corp: Employment, Equity Ownership. Agarwal:CTI BioPharma: Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V126.23.570.570

Language: English

Publisher: American Society of Hematology

Publication Date: 2015

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 120, No. 10 ( 2012-09-06), p. 1968-1969

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2012-07-442749

Language: English

Publisher: American Society of Hematology

Publication Date: 2012

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 120, No. 13 ( 2012-09-27), p. 2658-2668

Abstract: Sequestration in the bone marrow niche may allow leukemic stem cells to evade exposure to drugs. Because the CXCR4/SDF-1 axis is an important mechanism of leukemic stem cell interaction with marrow stroma, we tested whether plerixafor, an antagonist of CXCR4, may dislodge chronic myeloid leukemia (CML) cells from the niche, sensitizing them to tyrosine kinase inhibitors. We initially treated mice with retrovirally induced CML-like disease with imatinib plus plerixafor. Plerixafor mobilized CXCR4+ cells, but no difference was observed in leukemia burden, possibly reflecting insufficient disease control by imatinib. In a second series of experiments, we tested the combination of plerixafor with dasatinib in the same as well as an attenuated CML model. Despite much improved leukemia control, plerixafor failed to reduce leukemia burden over dasatinib alone. In addition, mice receiving plerixafor had an increased incidence of neurologic symptoms in association with CNS infiltration by BCR-ABL–expressing cells. We conclude that plerixafor is ineffective in reducing leukemia burden in this model but promotes CNS infiltration. Beneficial effects of combining tyrosine kinase inhibitors with plerixafor may be observed in a situation of minimal residual disease, but caution is warranted when disease control is incomplete.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2011-05-355396

Language: English

Publisher: American Society of Hematology

Publication Date: 2012

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 1308-1308

Abstract: Abstract 1308 Background: Despite the great strides that have been made in the treatment of acute myeloid leukemia (AML), one-third of patients are resistant to treatment. In many cases, disease-causing genetic targets still need to be elucidated. Cytokine and growth factor receptors contribute to cancer pathogenesis by regulating various downstream signaling cascades, including tyrosine kinase driven pathways, which represent amenable therapeutic targets. However, additional investigation is needed to understand the functional and therapeutic implications of cytokine and growth factor receptors in cancer pathogenesis. We have developed a novel RNAi-based screen to uncover the functional and prognostic relevance of growth factor/cytokine receptors for leukemia pathogenesis. This assay enables us to discover novel mechanisms of leukemogenesis involving various non-kinase receptors. Importantly, we show proof-of-principle data where IL2Rγ is found to be essential for JAK3 mutant mediated leukemogenesis. Methods and Results: To identify non-kinase cytokine and growth factor receptors that are critical for leukemia cell viability, we have designed a novel RNAi based functional screen targeting 188 growth factor receptors that were found to be highly expressed by gene microarray analysis of primary AML patient specimens. We have tested 40 AML patient samples and 5 AML cell lines for dependence on these receptors by electroporating cells with 188 individual siRNAs. After culturing cells for four days, effects on cell viability were measured using an MTS assay. Candidate targets found in primary patient samples include CD24, NCOA4, IL2Rα, IL15Rα and IL2Rγ. In many cases, we found that these targets have genetic abnormalities ranging from splice variation (IL2Rα) to intron retention (IL15Rα). In a few cases, the receptor was found to serve as a scaffold for regulating downstream oncogenic signaling and thus contributes to oncogenesis. For instance, we identified IL2Rγ as a potential target in the JAK3 A572V mutation positive AML cell line (CMK). Validation experiments demonstrated that knockdown of IL2Rγ significantly reduces the viability of CMK cells (90% decrease) and abrogates phosphorylation of JAK3 and downstream signaling molecules, STAT5, MAPK and the pS6 ribosomal protein. In a converse experiment, overexpression of IL2Rγ enhanced JAK3 A572V mediated signaling and increased its transformation potential in a ligand-independent manner. Similarly, IL2Rγ overexpression also increased the oncogenic potential of other JAK3 mutants such as M511I, which have previously been identified in AML patients. Intriguingly, the absence of IL2Rγ in murine bone marrow completely abrogated the clonogenic potential of JAK3 A572V as compared to IL2Rγ-wild type marrow. These effects can be rescued by co-expressing IL2Rγ with JAK3 A572V but not by co-expressing IL2Rγ with JAK3 Y100C, a FERM domain inactivating mutation. Additionally, overexpression of IL2Rγ made JAK3 A572V cells less sensitive to the JAK family inhibitor, JAK Inhibitor I (IC50is increased from 150 nM to 625 nM). Mechanistically, IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 protein levels and phosphorylation. In fact, mutant but not wild type JAK3 increased the expression of IL2Rγ, indicating IL2Rγ contributes to constitutive JAK3 signaling through a feedback mechanism. Overall these results demonstrate an oncogenic potentiating role of IL2Rγ. Conclusions: RNAi-based functional screening for AML cell dependence on non-kinase cytokine and growth factor receptors led to the identification of novel oncogenic etiologies for AML. These findings underscore the importance of cytokine and growth factor receptors in leukemia pathogenesis. This assay can identify genes that are crucial for malignant cell growth, regardless of the mutational status both in cell lines and in primary samples. Future studies integrating this RNAi screen with deep sequencing will lend additional power to this assay by accelerating our understanding of the genetic mechanisms underlying these functional gene targets such that these findings can be rapidly translated into novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V120.21.1308.1308

Language: English

Publisher: American Society of Hematology

Publication Date: 2012

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 3248-3248

Abstract: The ability to assess anti-leukemic activity of drugs on patient samples is a powerful tool in determining potential drug targets and may enhance selection of therapeutic agents with a biologic and functional rationale for individual patients. We have previously optimized small-molecule screens performed on freshly isolated leukemia cells for this purpose. Here we describe a novel method we have recently developed that produces reliable functional testing results from previously frozen specimens. This method was established to take advantage of bio-repositories containing archival primary leukemia specimens and to aid in the validation of potential drug target analysis and pathway activation. Three different culture media comprising various cocktails of cytokines were tested for their ability to maintain cell viability after thawing and to produce inhibitor results similar to those obtained using freshly isolated acute myeloid leukemia (AML) cells. We identified a method of thawing cells by first culturing for 24-48 hours in cytokine-enriched medium (RPMI-1640, 10% fetal bovine serum (FBS), and low concentrations of GM-CSF, G-CSF, SCF, and IL-3) prior to plating in RPMI-1640 and 10% FBS (R10). This method maintained high cell viability and produced inhibitor results comparable to those of freshly isolated cells plated in R10. Half-maximal inhibitory concentration (IC50) analysis was used to determine the relative success of the thaw process and reliability of results. Comparison of data produced from frozen cells initially cultured in cytokine-enriched medium to data obtained from freshly isolated cells showed few differences in effective drug hits. To examine the effects of freeze-thawing and cytokines on cell subpopulations, we performed FACS analysis on 8 markers to examine the level of cell differentiation under each condition. FACS sorting revealed increases in expression of myeloid differentiation markers (CD11b and CD14) after thaw in all media tested compared to fresh cells in cultured in R10. Thawed cells grown in cytokine-enriched medium remained closest to fresh controls as measured by antigen expression, including CD11b and CD14. Cell viability post-thaw was a strong indicator of samples that would fail to produce reliable inhibitor results. We observed that post-thaw viability 〈 80% served as a strong indicator of unreliable inhibitor data, even if dead cells were removed prior to plating. Cells cultured in cytokine-enriched medium for 24-48 hours resulted in 11-33% higher viability prior to plating in R10. Percent median IC50 for specific inhibitors assayed on fresh and frozen samples showed similar trends in effective drugs (Fig. 1A). Discrepancies observed in fresh and frozen sample data may be due to inconsistent fresh sample data as we demonstrated that frozen samples were highly consistent compared to other frozen samples (Fig. 1B). Lower R2 values could be attributed to variability observed with non-effective drugs. Taken together, our data indicate that thawing leukemia specimens in cytokine-enriched media can allow for the generation of informative small-molecule inhibitor screening data and provide a novel method for making use of archived primary human leukemia specimens for further downstream functional analyses. Figure 1. Median IC50 Comparison. Median IC50 values are calculated from 〉 3000 samples IC50 values. Percent median is the percent a particular sample IC50 compares to the median of all samples of that disease character. Mean values are shown for inhibitors with replicates. R2 from raw IC50. (A) Comparison of effective drug hits on fresh and frozen primary peripheral blood AML samples (R2 = 0.8151). (B) Comparison of effective drug hits obtained on a freshly isolated AML bone marrow sample versus 2 matched frozen/thawed samples from peripheral blood. Fresh vs. frozen (R2 = 0.45); frozen vs. frozen drug (R2 =0.9999). Figure 1. Median IC50 Comparison. Median IC50 values are calculated from 〉 3000 samples IC50 values. Percent median is the percent a particular sample IC50 compares to the median of all samples of that disease character. Mean values are shown for inhibitors with replicates. R2 from raw IC50. (A) Comparison of effective drug hits on fresh and frozen primary peripheral blood AML samples (R2 = 0.8151). (B) Comparison of effective drug hits obtained on a freshly isolated AML bone marrow sample versus 2 matched frozen/thawed samples from peripheral blood. Fresh vs. frozen (R2 = 0.45); frozen vs. frozen drug (R2 =0.9999). Disclosures Agarwal: CTI BioPharma: Research Funding. Druker:Oregon Health & Science University: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Sage Bionetworks: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; ARIAD: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V126.23.3248.3248

Language: English

Publisher: American Society of Hematology

Publication Date: 2015

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 138, No. Supplement 1 ( 2021-11-05), p. 1301-1301

Abstract: The development of molecularly-targeted therapies to improve outcomes relative to chemotherapy for acute myeloid leukemia (AML) is impeded by the heterogeneity of genetic aberrations that contribute to disease. Among the multitude of biological mechanisms that lead to AML disease initiation and progression is dysregulation of cytokine signaling pathways, a hallmark of chronic inflammation, which contribute to the growth, survival, and differentiation state of AML cells. We have previously shown that IL-1β, a pro-inflammatory cytokine expressed by many cell types including macrophages and monocytes, stimulates proliferation of leukemic blasts independent of mutational status in primary AML samples via enhanced phosphorylation of p38α MAPK, an effect that can be blocked by IL-1 receptor knockdown or by pharmacologic inhibition (Carey 2017). Additionally, recent studies have shown sensitivity to the approved BCL2 inhibitor venetoclax in AML associates with undifferentiated leukemic cells (Pei 2020; Zhang 2018; Majumder 2020). Based on these associations, we evaluated the combination of doramapimod (DORA), a p38 MAPK inhibitor, with venetoclax (VEN) for potential enhanced sensitivity on primary AML cells. Ex vivo drug screening of primary AML patient samples (n=335) revealed significantly enhanced efficacy of VEN+DORA compared to either single agent (Nemenyi test; p & lt;0.0001). This broad sensitivity of the VEN+DORA combination was not significantly associated with an array of clinical, genetic, and mutational features in the patient samples tested, in contrast to single agents, particularly VEN. Analysis of blood cell differential counts of patient samples tested identified increased monocyte levels were significantly correlated with sensitivity to DORA and resistance to VEN as single agents (Spearman r = -0.3 and 0.6; p & lt;0.0001), associations that were not apparent with the combination. For patient samples with accompanying FAB differentiation state-based designations (n=108), sensitivities of the combination were similar across classifications of undifferentiated (M0/M1) through monocytic (M4/M5) acute leukemia. In contrast, single-agent VEN was significantly more sensitive in undifferentiated compared to monocytic specimens, whereas DORA sensitivity showed the reverse trend (though to a lesser degree). These differences in sensitivity were further validated by immunophenotyping data where available (n=105), which showed surface markers associated with resistance to VEN (CD11b, CD14, CD16, CD56, CD64, HLADR; Wilcoxon Rank Sum, p & lt;0.001 to p=0.007) or sensitivity to VEN (CD117; p=0.001) or DORA (CD14; HLADR; p=0.004). By contrast, none of these associations significantly distinguished sensitivity for the VEN+DORA combination. Expression levels of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were concordant with their respective drug sensitivities associated with FAB classification; that is, significantly higher levels of BCL2 in M0/M1 leukemias and MAPK14 in M4/M5 cases (Mann-Whitney test; p & lt;0.0001; n=145). Further dissection of transcriptomic and drug sensitivity data revealed strong correlation and gene set enrichment for DORA and VEN sensitivities with monocyte-like and progenitor-like signatures, respectively (n=225), for cell differentiation states previously described for AML (van Galen 2019), and these associations diminished for the combination treatment. Lastly, the VEN+DORA combination enhanced efficacy and synergistic inhibition was confirmed using human AML cell line models tested with a matrix of potential dose concentrations. Taken together, these findings suggest that exploiting distinct, complementary sensitivity profiles of targeted therapies with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers an opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML. Disclosures Druker: Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Recludix Pharma, Inc.: Consultancy; EnLiven: Consultancy, Research Funding; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Aileron: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Genentech: Research Funding; Takeda: Research Funding; Astrazeneca: Research Funding; Constellation: Research Funding; Agios: Research Funding; Petra: Research Funding; Incyte: Research Funding; Array: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2021-152043

Language: English

Publisher: American Society of Hematology

Publication Date: 2021

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7