RAPID siRNA Screen for Identification of Therapeutic Gene Targets in Patients with Hematologic Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3978-3978
Author(s):  
Jeffrey W Tyner ◽  
Marc Loriaux ◽  
Stephanie G Willis ◽  
Bill H Chang ◽  
Vincent T Bicocca ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Small Molecule ◽  
Therapeutic Intervention ◽  
Tyrosine Kinases ◽  
Research Funding ◽  
Hematologic Malignancies ◽  
Patient Specific ◽  
Functional Screening ◽  
Primary Cells ◽  
Gene Target

Abstract Abstract 3978 Poster Board III-914 A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific kinases as cancer targets has been a slow process. Inhibition of cancer-causing tyrosine kinases offers a promising avenue of therapy, however this strategy of targeted therapy will require a detailed understanding of the oncogenic targets in each cancer patient. Here, we present an RNAi-assisted protein target identification (RAPID) assay by which cells from leukemia patients are functionally screened with siRNA to determine tyrosine kinases that constitute amenable targets for therapeutic intervention. Combination of the RAPID screen with gene-specific therapeutic approaches promises to yield a powerful synthesis of methodologies by which cancer patients can be specifically treated on the basis of functionally diagnosed gene targets. Methods To detect gene targets necessary for viability of malignant cells, we screened primary cells from 150 patients with hematologic malignancies by electroporating siRNAs individually targeting each member of the tyrosine kinase gene family. Four days later, we measured cell viability and tabulated sensitivity to silencing of specific genes. Samples were also screened for sensitivity to small-molecule kinase inhibitors. The mechanism of oncogenesis was investigated for each positive result. Results In total, we have identified 40 patient-specific gene targets in primary leukemia samples. We demonstrate that siRNA screening can identify known oncogenic lesions such as K-RasG13D and JAK2V617F in primary cells from leukemia patients. The RAPID screen has also directed us towards a novel insertional mutation in the thrombopoietin receptor, MPL (1886InsGG). Additionally, we have detected FLT3 sensitivity in patients with FLT3-ITD and loss of heterozygosity, although not in FLT3-ITD heterozygous patients. Agreement between siRNA-sensitive gene targets and small-molecule inhibitor sensitivity profiles has been high. The mechanism of oncogenesis and its relation to the gene target has been established in select other samples with abnormalities including gene overexpression and patient-specific mis-splicing events. Conclusions We demonstrate that RNAi functional screening can determine sensitivity to individual genes in cells obtained directly from cancer patients. Thus, this technique offers the potential to match targeted therapies with patients in a personalized manner. Application of these technologies will enable efficient discovery of the genetic etiology of cancer as well as a means for gene-specific therapeutic intervention. Disclosures: Deininger: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Calistoga: Research Funding; Genzyme: Research Funding. Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding.

RNAi Screening of the Tyrosine Kinome Identifies Therapeutic Targets in Leukemia Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 758-758
Author(s):  
Jeffrey W Tyner ◽  
Marc Loriaux ◽  
Stephanie G Willis ◽  
Bill Chang ◽  
Vincent T Bicocca ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Tyrosine Kinases ◽  
White Blood Cells ◽  
Functional Assay ◽  
Functional Screening ◽  
Whole Genome ◽  
Primary Cells ◽  
Gene Target

Abstract A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific tyrosine kinases as cancer targets has been a slow process. In the near future, whole-genome sequencing will enable vast amounts of sequence data to be collected, however clinical application of this information will require a detailed understanding of the functional consequences of each sequence change. Here, we present an RNAi-assisted protein target identification (RAPID) assay by which cells from leukemia patients are functionally screened with siRNA to determine tyrosine kinases that constitute amenable targets for therapeutic intervention. These data have led to identification of novel oncogenic anomalies in cancer patients. Combination of the RAPID screen with whole-genome sequencing promises to yield a powerful synthesis of methodologies by which both functional targets and genetic lesions can be rapidly determined. Methods: To detect targets necessary for viability of malignant cells, we screened primary cells from 75 patients with AML, ALL, CMML, and other MPD as well as white blood cells from healthy individuals by electroporating siRNAs individually targeting each member of the tyrosine kinase family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. Where possible, results were confirmed by treating samples with small-molecule inhibitors with activity against the genes identified by the assay. In addition, the mechanism of oncogenesis was investigated for each positive result. Results: We demonstrate that siRNA screening can identify known oncogenic lesions such as K-RasG13D and JAK2V617F in primary cells from leukemia patients. The RAPID screen has also directed us towards a novel insertional mutation in the thrombopoietin receptor, MPL (1886InsGG). Additionally, we have detected FLT3 sensitivity in patients with FLT3-ITD and loss of heterozygosity, although not in FLT3-ITD heterozygous patients. In total, of 75 patients screened, this assay has yielded 25 cases that exhibit sensitivity to one or more tyrosine kinases. The mechanism of oncogenesis and its relation to the gene target has been established in select other samples with genetic abnormalities including evidence of chromosomal rearrangements as well as gene overexpression and mis-spicing events. Conclusions: We demonstrate that RNAi functional screening can determine sensitivity to individual tyrosine kinases in primary samples. Thus, this technique offers the potential to match specific therapies for targeted intervention with individual patients based on a functional assay. Additionally, in many cases, combination of the RAPID screen with whole-genome sequencing will enable efficient discovery of the genetic etiology of cancer.

scholarly journals Perceptions of Hospice and Transfusion Access Among Patients with Advanced Blood Cancers: Results from a Best-Worst Scaling Survey

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3020-3020
Author(s):  
Oreofe O. Odejide ◽  
Scott F. Huntington ◽  
Eleanor Shi ◽  
Kimberly S. Johnson ◽  
James A. Tulsky ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Research Funding ◽  
Utility Score ◽  
Hospice Care ◽  
Hematologic Malignancies ◽  
Respite Care ◽  
Blood Transfusions ◽  
Blood Cancer ◽  
Best Worst Scaling ◽  
Hospice Use

Abstract Background: Patients with hematologic malignancies have low rates of hospice use, and when they do enroll, they often do so in the last three days of life. While lack of access to transfusions in hospice has been posited to be a key barrier to hospice use for this population, data are lacking regarding the perception of patients with blood cancers about the importance of transfusion access compared to traditional hospice services. We sought to characterize the utility of transfusions and hospice services from the perspective of patients with advanced blood cancers, who would potentially be eligible for hospice care. Methods: In October 2020, we began a web-based survey of patients with hematologic malignancies recruited from the clinics of two large cancer centers. Eligible patients were at least 18 years of age, had at least two outpatient visits to the cancer center, and had a physician-estimated prognosis of six months or less based on their hematologic oncologist answering "no" to a modified "surprise" question (Hudson KE, JPM 2018): "would you be surprised if this patient died within the next six months?" A physician-estimated prognosis of ≤ 6 months was used for study inclusion as this is an eligibility criterion for hospice. The survey was developed through literature review, a series of patient/caregiver focus groups (n=27) and cognitive debriefing with blood cancer patients (n=5). The survey included a best-worst scaling (BWS) section to assess patients' perceptions of the utility of various services routinely provided in hospice (visiting nurse, case manager, home health aide, chaplain, social worker, respite care) as well as non-routine services (transfusion access, transportation, peer support, telemedicine through videoconferencing). We asked patients to imagine a program developed to support quality of life for patients with blood cancers just like them. We then presented them with 10 questions with different combinations of the services in groups of 4 and participants were asked to select the service they considered "most important" and "least important" in deciding whether to sign up for the program. The BWS questions were constructed using a near balanced incomplete block design (Louviere JJ, Patient 2010). We conducted a hierarchical Bayesian analysis to ascertain a standardized utility score assigned to each service, a measure of the average propensity to choose a service as most important more often than least important. Analyses were conducted using Sawtooth (Sawtooth Software, Inc). Results: As of July 2021, 102 patients had completed the survey (response rate: 64.6%). The median age of respondents was 71 years (IQR 63, 77). The majority of respondents were male, white, and married/living with partner (Table). The most common diagnosis was acute leukemia (38.2%). Patients considered access to blood transfusions to have the highest importance (Figure) with a standardized utility score of 21.1, 95% confidence interval [CI] [19.6-22.6], followed by telemedicine (19.4, 95% CI [17.8-20.9]), transportation to/from medical appointments (13.4, 95% CI 11.7-15.1), and visiting nurses (10.8, 95% [9.5-12.2]. The three least important services perceived by respondents were access to respite care (4.7, 95% CI [3.9-5.6]), social workers (4.5, 95% CI [3.5-5.5]), and chaplains (2.3, 95% CI [1.3-3.3]). Conclusions: In this cohort of blood cancer patients who would potentially be eligible for hospice care, access to blood transfusions had the greatest level of importance relative to services routinely provided in hospice settings. The high value placed on transfusion access suggests that this factor plays a crucial role in hospice decision making. Accordingly, lack of transfusion access in many hospices likely reduces the propensity of patients with blood cancers to choose hospice. Innovative hospice delivery models that include access to palliative transfusions may have strong potential to increase hospice use and optimize end-of-life care for this patient population. Figure 1 Figure 1. Disclosures Huntington: Genentech: Consultancy; SeaGen: Consultancy; Thyme Inc: Consultancy; Servier: Consultancy; Novartis: Consultancy; Bayer: Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy; DTRM Biopharm: Research Funding; TG Therapeutics: Research Funding; Flatiron Health Inc.: Consultancy; Pharmacyclics: Consultancy, Honoraria; Celgene: Consultancy, Research Funding.

scholarly journals Financial Hardship Amongst Patients with Hematologic Malignancies: Using the EMR to Streamline and Prioritize Patient-Centered Care

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 661-661
Author(s):  
Sandeep S Voleti ◽  
Nandita Khera ◽  
Carolyn Mead-Harvey ◽  
Sikander Ailawadhi ◽  
Rafael Fonseca ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cancer Patients ◽  
Mayo Clinic ◽  
Research Funding ◽  
Hematologic Malignancy ◽  
Hematologic Malignancies ◽  
Cancer Center ◽  
Patient Centered ◽  
Advisory Committees ◽  
Alaskan Native

Abstract Background: Self-reported financial hardship (FH) amongst cancer patients is increasingly becoming a challenge for patients, caregivers, and healthcare providers. FH not only leads to financial struggles, significant lifestyle changes, and emotional distress, but also contributes to treatment noncompliance, affecting clinical outcomes. As treatment costs rise, it is crucial to develop efficient methods to proactively identify and alleviate FH in hematology practice. One potential approach is utilizing automated processes to identify those at highest risk of FH. At Mayo Clinic, screening for FH involves using a single financial strain question 'How hard is it for you to pay for the very basics like food, housing, medical care, and heating?' which all cancer patients answer annually as part of the institution's Social Determinants of Health (SDOH) assessment. Answers are on a five-point scale including not hard at all, not very hard, somewhat hard, hard, and very hard. In this study, we assess the prevalence and predictors for FH (denoted by a response of "Very hard" "Hard" or "Somewhat hard") amongst the Mayo Clinic hematologic malignancy patient population. Our study objective was to determine if this automated process could identify those at risk for FH. Methods: Patients who received care for hematologic malignancies (lymphoma, leukemia, plasma disorders, myelodysplastic/myeloproliferative disorders, and other heme malignancies) at any of the Mayo Clinic cancer centers (Minnesota, Arizona, and Florida) and who had completed the SDOH screen at least once were included in this study. The electronic medical record (EMR) and Mayo Clinic Cancer Registry were utilized to extract demographic and disease variables. Patient's home zip code was used to determine rural/urban residence, distance from cancer center, and the Area Deprivation Index (ADI), a measure of socioeconomic disadvantage based on home zip code (ranging from 1-100, with 100 representing the most disadvantaged). Multivariable logistic regression modeling was used to examine predictor variables for FH in this patient population. Results: The final cohort included 10,024 patients from 2018 to 2020. Median age was 64.6 years (IQR 58.1,73.7), 58% were male, and 79% married. Race/ethnicity composition was 94% White (n=9,268), 2.5% Black (n=246), 0.4% American Indian/Alaskan Native (44), and 4% Hispanic (n=360). Fifty-six percent of patients had Medicare and 41% had commercial insurance. Fifty percent were retired, 40% were working/students, and 72% were urban residents. Mean ADI was 41.2. Fifty-six percent of patients had lymphomas, 23.5% had plasma cell disorders, 8.5% had leukemias, 6.8% had other hematological malignancies, and 5.5% had myelodysplastic/myeloproliferative neoplasms. FH was reported by 12.8% (n=1286) of the patients. Table 1 shows the results of the multivariable model. A significantly higher likelihood of endorsing FH was noted in Hispanic vs non-Hispanics, Black and American Indian/Alaskan Native groups vs whites, Disabled/Unemployed vs working, Medicaid, Medicare, and Self-Pay groups vs commercial insurance, higher ADI (5 th quintile vs 1 st), and myelodysplastic/myeloproliferative disorder and other hematologic malignancy vs lymphoma patients. Older age, being retired, and living farther from the cancer center were associated with significantly less likelihood of endorsing FH. Conclusion: Our study used automated data extraction from the EMR to efficiently identify predictors of FH in hematologic cancer patients. Employing a dichotomized and automated "flag" for FH, particularly if incorporated in the EMR, could ease the identification of SDOH issues, facilitate timely connection to appropriate resources, and help provide better patient-centered care. Figure 1 Figure 1. Disclosures Ailawadhi: Sanofi: Consultancy; Cellectar: Research Funding; Karyopharm: Consultancy; Ascentage: Research Funding; Genentech: Consultancy; Janssen: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Beigene: Consultancy; GSK: Consultancy, Research Funding; AbbVie: Consultancy; Medimmune: Research Funding; Pharmacyclics: Consultancy, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Xencor: Research Funding. Fonseca: OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; BMS: Consultancy; Mayo Clinic in Arizona: Current Employment; Aduro: Consultancy; AbbVie: Consultancy; GSK: Consultancy; Merck: Consultancy; Juno: Consultancy; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Novartis: Consultancy; Bayer: Consultancy; Celgene: Consultancy; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy. Griffin: Exact Sciences: Research Funding.

Small Molecule Inhibitors of HER2 Inhibit Proliferative Signaling and Promote Apoptosis in Ph+ ALL

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1397-1397
Author(s):  
Mary E Irwin ◽  
Laura Nelson ◽  
Janice M Santiago-O'Farrill ◽  
Claudia P Miller ◽  
Doris R. Siwak ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Small Molecule ◽  
Tyrosine Kinases ◽  
Research Funding ◽  
Receptor Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Her2 Receptor ◽  
Small Molecule Kinase Inhibitor ◽  
Her2 Signaling

Abstract Abstract 1397 The ERBB family of receptor tyrosine kinases (EGFR, Her-2, Her-3 and Her-4) are receptor tyrosine kinases that, through mutation or aberrant expression, serve as oncogenes by promoting hallmark behaviors of cancer in many solid tumors. Previous work has suggested that HER2 is expressed in as much as 30% of B-ALL patients, and correlates with chemoresistance. We therefore hypothesized that HER2 signaling in Ph+ ALL may augment growth signaling and promote other malignant behaviors, such as resistance to cell death and independence from growth factors. Western blot and flow cytometric analyses of two human Ph+ ALL cell lines, Z119 and Z181, revealed cell surface expression of HER2, but not other family members. To determine the role of HER2 signaling in Ph+ ALL cell lines, the pan-HER family small molecule kinase inhibitor canertinib was used, and reverse phase protein array (RPPA) was conducted in Z119 and Z181 cell lines. Briefly, lysates from canertinib treated cells were spotted using a GeneTAC™ G3 arrayer onto nitrocellulose-coated FAST® slides. Incubation of the slides was performed with forty-three antibodies directed towards various cell signaling proteins followed by colorimetric detection and results were subsequently validated by western blotting. RPPA analyses revealed that treatment with canertinib effectively diminished HER2 phosphorylation in both cell lines. Additionally, we found decreased phosphorylation of the pro-survival molecules ribosomal protein S6, p70S6kinase, and c-Src, as well as increased expression of the pro-apoptotic molecules BIM and cleaved-PARP in both Ph+ ALL cell lines. Congruent with these findings, elevated activity of the executioner caspase 3 and increased DNA fragmentation, two distinct biochemical markers of apoptosis, were present after canertinib treatment in Z181 and Z119 cells, suggesting that inhibition of HER2 signaling results in programmed cell death of Ph+ ALL cell lines. This induction of apoptosis paralleled a decrease in overall proliferation of these cell lines, further implicating HER2 signaling in proliferation of Ph+ ALL. Next, we analyzed if clinically approved inhibitors of HER2 function could be utilized to produce the same biological consequence as canertinib in Ph+ ALL cell lines. Lapatinib (Tykerb) is a dual EGFR/HER2 small molecule kinase inhibitor approved by the FDA for the treatment of breast cancer. Consistent with our results utilizing canertinib, lapatinib was capable of inhibiting proliferation of both Z119 and Z181 cell lines. Interestingly, the FDA approved monoclonal antibody HER2 inhibitor trastuzumab (Herceptin) did not inhibit proliferation of these cell lines. Similarly, trimerized herceptin conjugates, which improve internalization of HER2 receptor, also had no effect on Ph+ ALL cell line proliferation. These results highlight an important distinction between the effects of the intracellular small molecule inhibitors of HER2 and monoclonal HER2 antibodies. In particular, extracellular engagement of the HER2 receptor by monoclonal antibodies may not be effective in targeting the HER2 signaling pathways required for proliferation and survival of Ph+ ALL. Taken together, our studies suggest that HER2 may play an important role in growth and survival signaling of Ph+ ALL cell lines and inhibition of HER2 with small molecule kinase inhibitors may improve treatment regimens. Thus, additional studies are warranted to determine the importance of HER2 in clinical specimens and the potential benefit of combining HER2 inhibitor therapy with imatinib treatment for Ph+ ALL. Disclosures: Mills: Glaxosmithkline: Research Funding; Pfizer: Research Funding.

Pharmacologic Thromboprophylaxis Is Frequently Prescribed In Hospitalized Cancer Patients At Academic Medical Centers: A Prospective, Cross-Sectional, Multi-Center Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2374-2374
Author(s):  
Adam A. Rojan ◽  
Nadia Q. Rehman ◽  
Renee E. Funches ◽  
Federico Campigotto ◽  
Jonathan Webster ◽  
...  
Keyword(s):  
Risk Factors ◽  
High Risk ◽  
Cancer Patients ◽  
Research Funding ◽  
Medical Center ◽  
Hematologic Malignancies ◽  
Cross Sectional ◽  
Medical Centers ◽  
Pharmacologic Thromboprophylaxis ◽  
Multi Center Study

Abstract Background Venous thromboembolism (VTE) is a frequent complication in hospitalized cancer patients and is associated with increased morbidity and mortality. Guidelines from major organizations recommend that all hospitalized patients with active malignancy receive pharmacologic thromboprophylaxis in the absence of bleeding or another contraindication. Nevertheless, reported rates of thromboprophylaxis use in hospitalized cancer patients have been low in several retrospective studies. We conducted a prospective cross-sectional study of hospitalized cancer patients at five academic hospitals to determine rates of thromboprophylaxis use and factors influencing the decision to administer thromboprophylaxis during hospitalization. Methods Administration of thromboprophylaxis to hospitalized cancer patients was assessed prospectively over consecutive days at five medical centers: University of Rochester, Johns Hopkins University, Beth Israel Deaconess Medical Center, University of California at Davis, and the DC Veterans Administration Medical Center/George Washington University. Data collected included reason for admission, cancer type and stage, and treatment as well as established risk factors for VTE including elements of the Padua Scoring System (PSS). The American College of Chest Physicians recommends the utilization of the PSS to guide thromboprophylaxis of hospitalized patients and a score of ≥4 is considered high risk for VTE. Univariate analysis for association of risk factors with the use of pharmacologic thromboprophylaxis was performed with two-sided Fisher exact tests and univariate logistic regression. Multivariable stepwise logistic regression model was performed to assess the influence of risk factors on the probability of receiving pharmacologic thromboprophylaxis. Results Seven-hundred and seventy-five patients were included in the study with a mean age of 56.3 years. Four hundred and thirty-five patients were male (56%) and 423 had hematologic malignancies (55%). The primary reason for admission was for cancer therapy in 254 cases (33%). Five hundred and eighty patients were considered high risk for VTE (≥4) using the PSS. Pharmacologic thromboprophylaxis was prescribed in 392 patients (51%, range 29%-71%). Accounting for contraindications to anticoagulation, 74% (N=528) of all cancer patients received appropriate hospital thromboprophylaxis. Among the cancer patients without contraindications for anticoagulation, individuals hospitalized with solid tumors were significantly more likely to receive thromboprophylaxis than those with hematologic malignancies (OR 2.34, 95% CI 1.43-3.82, P=0.0007). Cancer patients admitted for cancer-directed therapy (i.e. chemotherapy or radiation) were significantly less likely to receive thromboprophylaxis than those admitted for other medical conditions (OR 0.37 95% CI 0.22-0.61, P<0.0001). Sixty-three percent of low risk cancer patients as determined by PSS received anticoagulant thromboprophylaxis. Contraindications for anticoagulation were evident for the majority of the 383 patients (N=247, 64%) who did not receive pharmacologic thromboprophylaxis such as 161 with severe thrombocytopenia (42%), 43 with active hemorrhage (11%), 15 with a history of hemorrhage (4%), 2 with heparin induced thrombocytopenia (0.5%), and 11 on comfort-measure-only care (3%). Among the 136 patients who did not receive anticoagulation, 58.8% were considered high risk by the PSS. Conclusions This prospective, cross-sectional, multi-center study demonstrated that appropriate pharmacologic thromboprophylaxis is administered to the majority of hospitalized cancer patients. Despite absence of established benefit, the majority of lower risk cancer patients receive thromboprophylaxis during hospitalization. Disclosures: Wun: Daiichi-Sankyo: Research Funding. Rickles:Leo: Research Funding. Streiff:Bristol Myers Squibb: Research Funding; Sanofi: Consultancy, Honoraria; Eisai, Daiichi-Sankyo, Boehringer-Ingelheim, Janssen HealthCare: Consultancy. Khorana:Leo, Sanofi: Research Funding. Zwicker:Sanofi: Research Funding.

p38MAPK Inhibition Blocks Inflammatory Signaling in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2603-2603 ◽  
Author(s):  
Alyssa Carey ◽  
Swati Garg ◽  
Megan M Cleary ◽  
David K Edwards ◽  
Marc Loriaux ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Hematologic Malignancies ◽  
Sensitivity Profile ◽  
Acute Myeloid

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.

Preclinical Evaluation of AT219, a Small Molecule Inhibitor of MDM2 As an Anti-Myeloma Agent.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2948-2948
Author(s):  
Vijay G. Ramakrishnan ◽  
Teresa K. Kimlinger ◽  
Timothy Halling ◽  
Jessica Haug ◽  
Utkarsh Painuly ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Primary Cells ◽  
Wild Type ◽  
Drug Induced ◽  
Molecule Inhibitor ◽  
Wild Type P53 ◽  
Stock Solutions

Abstract Abstract 2948 Background: Deletions and mutations in the tumor suppressor protein p53 are an uncommon observation in new multiple myeloma (MM) patients and are observed more commonly in patients with advanced disease. p53 deletion has been observed to correlate with poor overall and progression free survival in MM patients. Wild type p53 modulates the expression levels of a broad array of proteins involved in cell cycle progression, apoptosis ultimately leading to cell cycle arrest and apoptosis. p53 is negatively regulated by MDM2. MDM2 binds to and ubiquitinates p53 marking it for proteasomal degradation. In addition, MDM2 is a direct downstream regulator of p53. Targeting the p53-MDM2 interaction by developing agents that bind to the p53 binding motif of MDM2 and reactivating p53 has therefore been an active area of research. Here, we present results from our pre-clinical studies using AT219, a small molecule inhibitor that binds to MDM2 preventing its interaction with p53. Methods: AT219 was obtained from Ascenta Therapeutics. Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Akt1/2 kinase inhibitor (Akti) was purchased from Sigma. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Mitocapture and cytochrome-c assays were also performed to confirm the induction of apoptosis in MM cell lines. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various time points. Results: AT219 induced potent cytotoxicity in MM cell lines MM1S, MM1R and H929, all three expressing wild type p53 with IC50 values of 2.5–5μM. Similar effects were observed when the above mentioned cell lines were treated with AT219 and the inhibitory effect of proliferation of these cells were examined. When MM1S or H929 cells were cultured with bone marrow stromal cells (BMSCs) derived from MM patients or with one of the three tumor promoting cytokines implicated in MM (IL6, IGF or VEGF) and treated with AT219, the drug was able to inhibit the proliferation of both cell lines to similar extents as observed when cultured independently without BMSCs or the cytokines. The increase in cytotoxicity was found to be due to cells undergoing apoptosis as observed when MM1S or H929 cells were cultured with AT219 and % apoptotic cells were measured as measured by annexin/PI, mitocapture and cytochrome c assays. AT219 was also observed to induce more potent apoptosis in primary cells obtained from new MM patients with wild type p53 than in cells obtained from relapsed MM patients with wild type p53. AT219 clearly upregulated p53 as observed by performing immunoblots after treatment with the drug in MM1S and H929 cells. In addition, MDM2 and p21 were also found to be significantly upregulated and Bax was slightly upregulated post drug treatment. Bcl2, Mcl1 and Xiap levels were down regulated. In MM1S cells AT219 treatment resulted in a slight down regulation of pAkt (Ser 473). However, in H929 cells we observed a transient upregulation of pAkt following AT219 treatment. This prompted us to test AT219 in combination with Akti on MM cell lines. Our results on both MM1S and H929 cells using AT219 in combination with Akti demonstrated synergy. We are currently testing this combination in primary cells drawn from MM patients with both wild type p53 and those with p53 deletions and mutations. Conclusions: Our studies validate the anti-MM activity of AT219 in MM patients with wild type p53. In addition to using AT219 in combination with Akti, we are testing AT219 in combination with existing anti- MM chemotherapeutic agents. Interesting results from our studies will form the basis for clinical evaluation of AT219 as a single agent or in combination with an Akt inhibitor or other agents in MM patients. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

Palliative Care For Patients With Hematologic Malignancies: A Profile Of Patients With Blood Cancers Referred To The Choice Hospice Network

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1678-1678 ◽  
Author(s):  
Thomas W. LeBlanc ◽  
Amy P Abernethy ◽  
David J Casarett
Keyword(s):  
Palliative Care ◽  
Length Of Stay ◽  
Board Of Directors ◽  
Cancer Patients ◽  
Research Funding ◽  
Hematologic Malignancies ◽  
Care Needs ◽  
Advisory Committees ◽  
Time Of Admission ◽  
Palliative Care Needs

Abstract Background Published literature provides an incomplete description of the palliative care needs of the hematologic malignancies (HM) population. We sought to compare characteristics of hospice patients with HM vs. those with solid tumors. Methods Patient data (2008-2012) were extracted from the electronic records of 10 hospices in the CHOICE network (Coalition of Hospices Organized to Investigate Comparative Effectiveness). We identified the subset with HM using ICD-9 codes, comparing them to the subset with all other neoplasias using bivariate analyses. Results Of the 48,147 cancer patients, 3,518 (7.3%) had a hematologic malignancy, with rates not changing substantially over the 5-year study period (Table 1). HM patients were somewhat older and more often male, with a slight, but significant tendency to be more often Caucasian (see Table 1). At time of admission, HM patients had worse physical functioning by Palliative Performance Scale (PPS) scores (32% vs. 24% were below 40; p<0.001) and were more likely to be admitted to an inpatient hospice or nursing home setting vs. home (OR 1.34, CI 1.16-1.56, and OR 1.54, CI 1.39-1.72; p<0.001). HM patients were more likely to have a hospice length of stay (LOS) of 1 day (OR 1.66, CI 1.49-1.86; p<0.001), or of less than 7 days (OR 1.68, CI 1.56-1.81; p<0.001). Patients with leukemia were more likely to have a LOS of 1 day compared to those with lymphoma (OR 1.31, CI 1.1-1.56; p=0.002), and were more likely to die during the study period (HR 1.23, CI 1.13-1.34; p<0.001). Patients with multiple myeloma or myelodysplastic syndrome were more likely than other HM patients to be taking an opioid at admission (OR 1.37, CI 1.12-1.67; p=0.002, and OR 1.29, CI 1.01-1.64; p =0.042). Patients with myelodysplasia were also more likely to have an advance directive or durable power of attorney in place at admission compared to other HM patients (OR 1.36, CI 1.07-1.72; p=0.012). Conclusions Patients with hematologic malignancies who are referred to hospice appear to be more seriously ill at time of admission, with worse physical functioning scores by PPS and shorter length of stay compared to other cancer patients. Targeted efforts to better understand the palliative care needs of the hematologic malignancies population are warranted. Disclosures: Abernethy: American Academy of Hospice and Palliative Medicine: Membership on an entity’s Board of Directors or advisory committees; Biovex: Research Funding; DARA: Research Funding; Helsinn: Consultancy, Research Funding; MiCo: Research Funding; Dendreon: Research Funding; Pfizer: Consultancy, Research Funding; Alexion: Research Funding; Amgen: Research Funding; Genentech: Research Funding; Bristol Myers Squibb: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Consultancy; Advoset: Membership on an entity’s Board of Directors or advisory committees; Orange Leaf Associates, LLC: Membership on an entity’s Board of Directors or advisory committees.

Hematologic and Hospitalization Outcomes in Patients with Chemotherapy-Induced Anemia (CIA) Treated with Erythropoiesis-Stimulating Agents (ESAs) in the Pre- Vs Post-National Coverage Determination (NCD) Time Periods.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2482-2482
Author(s):  
Elizabeth Apgar ◽  
Tanya M. Burton ◽  
Kay Larholt ◽  
Chris L. Pashos ◽  
Lorie Ellis ◽  
...  
Keyword(s):  
Blood Transfusion ◽  
Cancer Patients ◽  
Research Funding ◽  
Study Patient ◽  
Patient Specific ◽  
Tumor Type ◽  
Ortho Biotech ◽  
Blood Utilization ◽  
Time Periods ◽  
Hospital Days

Abstract Abstract 2482 Poster Board II-459 Background: National coverage limitations for ESA treatment in cancer patients with CIA were established by the Centers for Medicare & Medicaid Services in July 2007. Clinical outcomes based on ESA dosing described in the NCD have not been reported in prospective observational or clinical trial data. To understand hematologic and hospitalization outcomes in CIA patients treated in Pre- and Post-NCD time periods, an analysis of data from the D.O.S.E. (Dosing and Outcomes Study of Erythropoiesis-Stimulating Therapies) registry, an ongoing prospective observational study, was conducted. Methods: ESA-treated cancer patients with CIA were selected if they initiated ESA treatment 12/01/2005-04/01/2007 (Pre-NCD) or 10/01/2007-02/01/2009 (Post-NCD), had ≥ 2 ESA administrations, and had both a baseline and ≥ 1 transfusion-independent post-ESA initiation hemoglobin (Hb) assessment. Assessed outcomes included proportion of patients receiving blood transfusion, number of units/study patient, Hb levels (at baseline and at weeks 4, 8, 12, 16) and hospitalization rates (admissions, number of hospital days) adjusted for ESA therapeutic duration (defined as time (days) from first to last ESA administration + patient-specific ESA treatment interval). Results: This analysis included 836 patients (Pre-NCD 585; Post-NCD 251) from 54 sites. Patients in the Pre-NCD and Post-NCD cohorts were similar in gender distribution and weight. The Post-NCD cohort was significantly older (64.7 yrs vs. 61.9 yrs; p<0.01). The groups also differed significantly in overall tumor type distribution (p<0.0001). The Post-NCD group had a lower proportion of patients with breast cancer and higher proportion of patients with lung cancer and gynecologic malignancies. ESA treatment duration was significantly shorter in the Post-NCD group (mean days ± SD: Post-NCD 55.5 ± 32.7, Pre-NCD 65.8 ± 33.8, p<0.0001). The proportion of patients receiving blood transfusion was significantly greater in the Post-NCD group (Post-NCD 26.7%, Pre-NCD 15.6%, p=0.0002) as was blood utilization (Units/study patient: Post-NCD 0.9, Pre-NCD 0.4, p=0.0001). As shown in the table, Hb levels were significantly lower at all time points in the Post-NCD group. The rate of hospital admissions was significantly greater in the Post-NCD group [Admissions/100-patient ESA therapeutic days (95% CI): Post-NCD 0.42 (0.34, 0.53), Pre-NCD 0.28 (0.24, 0.33)] as was the number of hospital days [Hospital days/100-patient ESA therapeutic days (95% CI): Post-NCD 2.3 (2.1, 2.5), Pre-NCD 1.3 (1.2, 1.4)]. Conclusions: Significantly greater blood utilization and lower Hb levels were observed in ESA-treated CIA patients in the Post-NCD period compared to the Pre-NCD period. Rates of hospitalizations and hospital length of stay were also significantly greater in the Post-NCD group. Study of comparative hematologic and resource utilization outcomes before and after the NCD is warranted in additional clinical centers. Disclosures: Apgar: Centocor Ortho Biotech Services, LLC: Consultancy, Research Funding. Burton:Centocor Ortho Biotech Services, LLC: Consultancy, Research Funding. Larholt:Centocor Ortho Biotech Services, LLC: Consultancy, Research Funding. Pashos:Centocor Ortho Biotech Services, LLC: Consultancy, Research Funding. Ellis:Centocor Ortho Biotech Services, LLC: Employment. Senbetta:Centocor Ortho Biotech Services, LLC: Employment. McKenzie:Centocor Ortho Biotech Services, LLC: Employment.

CSF1R Inhibition Targets AML Cells By Depleting Supportive Microenvironmental Signal from CD14+ Monocytes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3824-3824 ◽  
Author(s):  
David K Edwards ◽  
Angela Rofelty ◽  
Anupriya Agarwal ◽  
Stephen E Kurtz ◽  
Elie Traer ◽  
...  
Keyword(s):  
Cell Viability ◽  
Board Of Directors ◽  
Small Molecule ◽  
Research Funding ◽  
Small Molecule Inhibitors ◽  
Functional Screening ◽  
Advisory Committees ◽  
Equity Ownership ◽  
The Impact ◽  
Dependent Mechanism

Abstract Introduction: CSF1R (M-CSFR) is a receptor tyrosine kinase that, upon binding with CSF1 ligand (M-CSF), activates downstream survival, proliferation, and differentiation pathways in myeloid lineage cells. Expression and activity of CSF1R has been shown to be important for sustaining tumor-associated macrophages (TAMs) in a variety of solid tumor types, and these TAMs have been shown to provide a supportive microenvironment that maintains tumor cell viability and growth. Monocyte and macrophage lineage cells have also been implicated in providing a supportive microenvironment in certain hematologic malignancies, such as nurse-like cells in CLL, but have not been extensively explored in AML. Using an ex vivo functional screening platform applied directly to primary specimens from AML patients, we have identified recurrent sensitivity to CSF1R inhibition or silencing due to a depletion of CSF1R-expressing monocytic-lineage cells in the AML microenvironment. Methods: To identify new therapies for AML patients, our lab has screened hundreds of AML patient specimens against panels of targeted small-molecule inhibitors or siRNA and evaluated the impact on cell viability. These panels have included two small-molecule inhibitors with exquisite specificity for CSF1R (GW-2580 and ARRY-382). We have also used a panel of recombinant growth factors and cytokines, including CSF1, to measure their impact on stimulating AML cell growth. Results: We found that 20-30% of AML specimens showed sensitivity to inhibition or silencing of CSF1R, with good concordance between CSF1R siRNA, GW-2580, and ARRY-382. Exhaustive analysis of the clinical, demographic, and genetic features of these patients failed to reveal correlation between CSF1R and any prominent disease subsets. Samples sensitive to CSF1R silencing or inhibition are significantly more likely than non-sensitive samples to respond to CSF1 growth stimulation, indicating a ligand-dependent mechanism of CSF1R signaling. Additionally, we examined CSF1R and CSF1 expression in discrete hematopoietic cell populations by flow cytometry in our specimens, both with and without CSF1R inhibition. Interestingly, we found that CSF1R was predominantly expressed a subpopulation of CD14-expressing monocytes, not tumor cells, and this population disappeared after exposure to the CSF1R inhibitor. Moreover, we identified a correlation between sensitivity to CSF1R inhibition and the presence of this CD14/CSF1R-expressing cell population. Conclusions: These results suggest that CD14/CSF1R-expressing cells support AML blasts through a ligand-dependent mechanism, and depleting these cells eliminates the supportive microenvironment, resulting in leukemia cell death. Therefore, we propose using CSF1R inhibitors, such as ARRY-382, as a promising new line of therapy to target AML by disrupting the tumor microenvironment. Disclosures Agarwal: CTI BioPharma: Research Funding. Lee:Array Biopharma: Employment. Chantry:Array Biopharma: Employment. Druker:Sage Bionetworks: Research Funding; Millipore: Patents & Royalties; Molecular MD: 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; Bristol-Myers Squibb: Research Funding; McGraw Hill: Patents & Royalties; CTI Biosciences, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; AstraZeneca: Consultancy; Oregon Health and Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; Novartis Pharamceuticals: Research Funding; ARIAD: Research Funding; Aptose Therapeutics Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Incyte: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Array Biopharma: Research Funding.

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