The outcome for pediatric acute lymphoblastic leukemia (ALL) patients who relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly ...glucocorticoids. In this study, we performed a genome-scale short hairpin RNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of these data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrate that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of the MEK1/2 target ERK in matched diagnosis-relapse primary samples and observed increased phosphorylated ERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Together, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents and that the MAPK pathway is an attractive target for prevention and/or treatment of relapsed disease.
•Genetic or pharmacologic inhibition of MEK4 and MEK2 enhances prednisolone-induced cell death in ALL models.•MAPK signaling cascades are activated at relapse compared to diagnosis in ALL samples and have enhanced response to MEK inhibition.
While some children with acute lymphoblastic leukemia (ALL) have excellent prognoses, the prognosis for adults and children with T cell ALL is more guarded. Treatment for T-ALL is heavily dependent ...upon antimetabolite chemotherapeutics, including cytarabine. Targeted inhibition of WEE1 with AZD1775 has emerged as a strategy to sensitize cancer cells to cytarabine and other chemotherapeutics. We sought to determine if this strategy would be effective for T-ALL with clinically relevant anti-leukemia agents. We found that AZD1775 sensitizes T-ALL cells to several traditional anti-leukemia agents, acting synergistically with cytarabine by enhancing DNA damage and apoptosis. In addition to increased phosphorylation of H2AX at serine 139 (γH2AX), AZD1775 led to increased phosphorylation of H2AX at tyrosine 142, a signaling event associated with promotion of apoptosis over DNA repair. In a xenograft model of T-ALL, the addition of AZD1775 to cytarabine slowed leukemia progression and prolonged survival. Inhibition of WEE1 with AZD1775 sensitizes T-ALL to several anti-leukemia agents, particularly cytarabine and that mechanistically, AZD1775 promotes apoptosis over DNA repair in cells treated with cytarabine. These data support the development of clinical trials including AZD1775 in combination with conventional chemotherapy for acute leukemia.
This study assessed whether myristoylated alanine-rich C-kinase substrate (MARCKS) can regulate glioblastoma multiforme (GBM) growth, radiation sensitivity, and clinical outcome.
MARCKS protein ...levels were analyzed in five GBM explant cell lines and eight patient-derived xenograft tumors by immunoblot, and these levels were correlated to proliferation rates and intracranial growth rates, respectively. Manipulation of MARCKS protein levels was assessed by lentiviral-mediated short hairpin RNA knockdown in the U251 cell line and MARCKS overexpression in the U87 cell line. The effect of manipulation of MARCKS on proliferation, radiation sensitivity, and senescence was assessed. MARCKS gene expression was correlated with survival outcomes in the Repository of Molecular Brain Neoplasia Data (REMBRANDT) Database and The Cancer Genome Atlas (TCGA).
MARCKS protein expression was inversely correlated with GBM proliferation and intracranial xenograft growth rates. Genetic silencing of MARCKS promoted GBM proliferation and radiation resistance, whereas MARCKS overexpression greatly reduced GBM growth potential and induced senescence. We found MARCKS gene expression to be directly correlated with survival in both the REMBRANDT and TCGA databases. Specifically, patients with high MARCKS expressing tumors of the proneural molecular subtype had significantly increased survival rates. This effect was most pronounced in tumors with unmethylated O(6)-methylguanine DNA methyltransferase (MGMT) promoters, a traditionally poor prognostic factor.
MARCKS levels impact GBM growth and radiation sensitivity. High MARCKS expressing GBM tumors are associated with improved survival, particularly with unmethylated MGMT promoters. These findings suggest the use of MARCKS as a novel target and biomarker for prognosis in the proneural subtype of GBM.
Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and ...salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone.
Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX).
Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone.
Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated.
No relevant conflicts of interest to declare.
Introduction: Geriatric assessment (GA) is a multidimensional evaluation of patient health and function that may detect impairments not identified as part of routine care, predict treatment-related ...morbidity and mortality, and inform treatment plans. Given evidence of these benefits, the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (NCCN Guidelines®) recommend GA for older candidates of hematopoietic stem cell transplantation (HCT). However, both older and younger HCT candidates will often receive multiple rounds of chemotherapy before HCT, leading to functional impairments in all age groups. Furthermore, HCT patients often experience a significant gap between when they are first evaluated and actually proceed to transplant (e.g., while a donor search is conducted), creating an opportunity to identify impairments and optimize function prior to transplant.
Methods: To address this opportunity, we created a clinical pre-HCT optimization program (C-POP) to evaluate physical function, cognitive function, nutritional status, and mental health in all adults who were deemed potential candidates for allogeneic HCT by a HCT physician. We applied this standard of care program to all adult candidates for HCT, regardless of age, with the goal of identifying functional impairments and then referring patients to services to optimize those impairments prior to HCT. We defined impairments using validated measures and compared results to established norms or scoring, controlling for age and gender where appropriate (e.g., the cut-off for six-minute walk distance was adjusted for age, gender, height, and weight, while the cut-off for falls was any fall regardless of characteristics). Patients with impairments were referred to the appropriate supportive care (e.g., physical function impairment -> referral to physical therapy). Results were prospectively analyzed at new patient evaluation (NPE), which was the first time the patient met a HCT physician and sign-off, which occurred within a week before starting transplant. While the program is ongoing, we present here the results of patients evaluated between October 16th, 2017 and July 1st, 2019. Patients are divided into three pre-specified age groups: <40 years old, 40-59 years old, and >=60 years old, with results compared using a chi-squared test.
Results: We evaluated 115 patients: 21 (18%) <40 years, 40 (35%) 40-59 years, and 54 (47%) >=60 years). There were no differences between the age groups in other demographics (gender, race, and ethnicity). At NPE, 93 (81%) met criteria for at least 1 impairment in physical function, cognitive function, nutritional status, or mental health; 62 (54%) met criteria for impairments in 2 or more areas. Surprisingly, patients <40 years were more likely to screen positive for physical function (20/21, 95%) than patients 40-59 years (26/40, 65%) and patients >=60 years (36/54, 67%) (p=0.03).
Of those 115 patients, 52 (45%) proceeded to HCT, including 12 (57%) <40 years, 18 (45%) 40-59 years, and 22 (41%) >=60 years (p=0.75); of those patients who have not proceeded to HCT, 40 (35%) will never proceed to HCT (e.g., deemed not a candidate after functional evaluation or died of disease prior to HCT) while 23 (20%) are still awaiting HCT (e.g., donor search ongoing). Patients who proceeded to HCT were less likely to have mental health impairments (2/52, 4% vs. 9/40, 23%, p=0.006).
Of the 52 who were seen at new patient evaluation and proceeded to transplant, 40 (77%) were seen at sign off. Of those who had impairments at NPE, 12/23 (52%) improved their physical function to normal limits, 4/9 (44%) improved their cognitive function, and 9/13 (69%) improved their nutritional status by the time of sign-off (of those who were seen at sign-off, none had mental health impairments at NPE).
Discussion: These results demonstrate that younger as well as older candidates for HCT exhibit a high degree of functional impairment. However, this impairment could be amenable to improvement prior to HCT. These findings support application of GA to all HCT candidates regardless of age. We will investigate the effect of referred interventions (e.g., physical therapy, seeing a dietician) in improving functional impairments in future studies, as well as look at the effect of these findings on HCT outcomes.
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Wiggins:Incyte, Inc.: Speakers Bureau. Gasparetto:Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Rizzieri:Millennium: Speakers Bureau; Novartis: Consultancy; AbbVie: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Incyte: Consultancy, Speakers Bureau; TEVA: Consultancy; Spectrum: Consultancy; Kite Pharma: Consultancy; Gilead Sciences: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau. Horwitz:Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Sung:Novartis: Research Funding; Merck: Research Funding; Seres: Research Funding.
Allogeneic hematopoietic stem cell transplantation (HCT) is a potentially curative treatment for both malignant and nonmalignant hematologic diseases; however, reported rates of treatment-related ...mortality approach 30%. Outcomes are worse in patients who begin HCT with functional impairments. To detect such impairments, a geriatric assessment (GA) is recommended in adults age ≥65 years. Younger HCT candidates also may be impaired because of chemotherapy regimens pre-HCT. Therefore, we hypothesized that GA can be beneficial for adult patients of all ages and subsequently created a clinical pretransplantation optimization program to assess all HCT candidates using a modified GA. One-hundred fifty-seven patients were evaluated in 4 functional domains- physical, cognitive, nutritional, and psychological-at 2 time points prior to HCT-new patient evaluation (NPE) and sign-off (SO)-between October 2017 and January 2020. At NPE, 80.9% of the patients had at least 1 domain with a functional impairment, and physical (P = .006), cognitive (P = .04), and psychological (P = .04) impairments were associated with an increased likelihood of not proceeding to HCT. In addition, patients age 18 to 39 years were more likely than older patients to have a physical function impairment (P = .001). Between NPE and SO, 51.9% of the patients had resolution of 1 or more impairments, and nutritional impairment at SO was predictive of worse overall survival (P = .01). Our study shows that GA can identify functional impairments in patients of all ages. Early identification of impairments could facilitate referrals to supportive care and resolution of impairments prior to HCT, suggesting that GA could be recommended for HCT candidates of all ages.
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