A multiparametric niche-like drug screening platform in acute myeloid leukemia.

Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular (NPM1c MRD, p = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.

P2RY2-AKT activation is a therapeutically actionable consequence of XPO1 inhibition in acute myeloid leukemia.

Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.

Cystine uptake inhibition potentiates front-line therapies in acute myeloid leukemia.

By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.

Targeting serine hydroxymethyltransferases 1 and 2 for T-cell acute lymphoblastic leukemia therapy.

Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.

Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

The Folate Cycle Enzyme MTHFR Is a Critical Regulator of Cell Response to MYC-Targeting Therapies.

Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid-related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme MTHFR, which exhibits reduced-function polymorphisms in about 10% of Caucasians, induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Furthermore, this effect is abrogated by supplementation with the MTHFR enzymatic product CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies. SIGNIFICANCE: Although MYC-targeting therapies represent a promising strategy for cancer treatment, evidence of predictors of sensitivity to these agents is limited. We pinpoint that folate cycle disturbance and frequent polymorphisms associated with reduced MTHFR activity promote resistance to BET inhibitors. CH3-THF supplementation thus represents a low-risk intervention to enhance their effects.See related commentary by Marando and Huntly, p. 1791.This article is highlighted in the In This Issue feature, p. 1775.

Milnacipran in provoked vestibulodynia: efficacy and predictors of treatment success.

OBJECTIVE: This study aimed to collect preliminary evidence on the efficacy of milnacipran in reducing pain in women with provoked vestibulodynia (PVD) and to identify which patient characteristics predict treatment success. MATERIALS AND METHODS: A 12-week open-label trial was conducted in 22 women with PVD. The Pain Rating Index of the McGill Pain Questionnaire was the primary outcome measure. Other outcome measures included daily diaries, Beck Depression Inventory, State-Trait Anxiety Inventory, Female Sexual Function Index, Brief Pain Inventory, a personal or family history of fibromyalgia, and PVD subtype. RESULTS: Milnacipran (50-200 mg/d) significantly reduced pain severity on the Pain Rating Index (p = .001), coital pain (p = .001), tampon pain (p = .003), and mean vulvar pain (p ≤ .001). Scores were also decreased on the Beck Depression Inventory (p = .015), State-Trait Anxiety Inventory (p = .046), and Brief Pain Inventory (p = .019) and increased on the Female Sexual Function Index (p = .004). Fibromyalgia history, PVD subtype, presence of depression or anxiety, and level of impairment did not affect treatment response. By logistic regression analysis, it was noted that the odds of treatment success was 3 times higher among women who, at pretreatment, had a sexually satisfying relationship compared to those who did not (odds ratio = 3.30, confidence interval = 1.04-10.50, p = .043). CONCLUSIONS: Milnacipran significantly reduced vestibular pain in women with PVD. Treatment success was predicted by pretreatment sexual satisfaction. A larger randomized controlled trial is necessary to confirm the efficacy of milnacipran in PVD and to identify other possible predictors of treatment outcome.

Guided outcomes in learned efficiency model in clinical medical education: a randomized controlled trial of self-regulated learning.

OBJECTIVE: The guided outcomes in learned efficiency (GOLE) model emphasizes the use of evidence-based resources to understand the diagnosis, treatment, follow-up, and prevention of disease. We seek to determine whether presentations created using the GOLE model are superior to an unstructured approach in achieving Accreditation Council for Graduate Medical Education (ACGME) Core Competencies. STUDY DESIGN: Consenting medical students were randomized to GOLE or control groups to individually research a self-selected clinical topic. A validated survey instrument was used prepresentation and postpresentation to assess perceived improvement in knowledge. Subjects completed self-evaluations at enrollment and after presentation of their chosen clinical topic. Other students, residents, and a faculty member also completed evaluations after each student presentation. Standard statistical methods (analysis of variance, 2-tailed t test) were used to determine if a statistically significant difference existed between intervention and control groups. RESULTS: Self-assessments were similar in the GOLE and control groups. Externally perceived presentation scores were greater in the GOLE group (ACGME global P < .0001, presentation global P = .07), which demonstrated a significant improvement in 5 core competencies. Time spent preparing the presentation and resources utilized did not differ between groups. CONCLUSION: The presentations prepared using the GOLE model were rated higher by observers than those prepared using traditional techniques.

Citalopram in the treatment of women with chronic pelvic pain: an open-label trial.

OBJECTIVE: To determine the efficacy of citalopram in the treatment of chronic pelvic pain by measuring changes in pain severity, depressive symptoms and functional disability. STUDY DESIGN: Fourteen women between 18 and 50 years of age with chronic pelvic pain were enrolled in a 12-week, open-label, flexible-dose study. Following a single-blind washout, placebo nonresponders were treated with citalopram (20-60 mg/d). RESULTS: Twelve patients completed the study. Depression scores decreased significantly on the Hamilton Psychiatric Rating Scale for Depression (p = 0.006), pain severity showed a trend toward improvement on the McGill Pain Intensity Scale (p = 0.096), but there was no significant differences on the Pain Disability Index (p = 0.158). Eleven of 12 (91.7%) patients elected to continue taking citalopram after study completion. CONCLUSION: Citalopram is effective in reducing depressive symptoms, shows a statistical trend toward improvement in pain intensity in women with chronic pelvic pain and is well tolerated. It appears minimally effective in reducing disability. Larger, controlled studies are needed to evaluate the role of citalopram in treating chronic pelvic pain.

A comparison of an interactive computer-based method with a conventional reading approach for learning pelvic anatomy.

OBJECTIVE: This study was undertaken to assess the impact of interactive, computer-based versus conventional, paper-based format in student, resident, and fellow learning and retention of anatomy knowledge. STUDY DESIGN: Randomized longitudinal cohort design with scores repeated as pre-, post-, and follow-up tests. Subjects were randomly assigned to an anatomy module in computer-based (CD-ROM) format and 1 in paper-based format. A follow-up examination was administered 3 weeks after the posttest to evaluate retention of knowledge. Tests results were analyzed by using Student t tests and analysis of variance. RESULTS: Thirty-nine subjects completed all testing. Regardless of instructional method, pretest to posttest scores improved (P < .01), and posttest to follow-up test scores decreased among all levels of training (P < .01). Student satisfaction was highest with CD-ROM format. CONCLUSION: Improvement and retention of anatomy knowledge was not significantly different when comparing a new CD-ROM interactive approach with a traditional paper-based method.

Assessment of a 360-degree instrument to evaluate residents' competency in interpersonal and communication skills.

OBJECTIVE: To test the reliability of the 360-degree evaluation instrument for assessing residents' competency in interpersonal and communication skills. METHOD: Ten-item questionnaires were distributed to residents and evaluators at Monmouth Medical Center in Long Branch, New Jersey, in March/April, 2002. The scoring scale was 1-5; the highest score was 50. Data were maintained strictly confidential; each resident was assigned a code. Completed data sheets were collated by category and entered into a spreadsheet. The total and mean scores by each category of evaluator were calculated for each resident and a rank order list created. Shrout-Fleiss (model 2) intraclass correlation coefficients measured reliability of ratings within each group of evaluators. Reliability/reproducibility among evaluators' scores were tested by the Pearson correlation coefficient (p <.05). RESULTS: Intraclass correlation coefficients showed a narrow range, from.85-.54. The highest ranked resident overall ranked high and the lowest was low with most evaluators. The rank order among fellow residents was markedly different from other evaluator categories. Pearson correlation coefficients showed significant correlation between faculty and ancillary staff, (p =.002). Patients as evaluators did show intraclass correlation, but did not correlate significantly with other categories. Scores from colleagues correlated negatively with all other categories of evaluators. CONCLUSIONS: The 360-degree instrument appears to be reliable to evaluate residents' competency in interpersonal and communication skills. Information from the assessment may provide feedback to residents. Areas of improvement identified by the scores would suggest areas for improvement and further ongoing assessment.

Pap smear noncompliance among female obstetrics-gynecology residents.

OBJECTIVES: We wish to determine how well female obstetrics and gynecology (OB/GYN) residents in the United States follow recommendations for routine Pap smears in managing their own health care and to identify the reasons for noncompliance. METHODS: A survey was mailed to 1693 female OB/GYN residents in the U.S. Factors associated with noncompliance were determined by chi-square. Logistic regression analysis was used to identify independent prognostic factors and calculate the odds ratio (OR). RESULTS: Six hundred eleven (36%) surveys were returned with compliance information. Noncompliance was reported by 33%. Lack of time or inconvenience was cited as the most common reason (93%) for noncompliance, followed by consideration of themselves as low risk for cervical disease (41%) and fear of or embarrassment in seeing a doctor (14%). On multivariate analysis, independent risk factors for noncompliance during residency included noncompliance prior to residency (OR 4.6, P<0.0001) and Asian ethnicity (OR 2.1, P = 0.02), whereas East Indian ethnicity (OR 3.0, P = 0.06) and having no children (OR 1.6, P = 0.07) were near significant. CONCLUSIONS: A substantial proportion of female OB/GYN residents do not follow standard recommendations for routine Pap smears. Although the principal reason for noncompliance given was lack of available time and/or inconvenience during residency, this behavior predated residency and was associated with specific ethnic groups.

Chiral recognition: design and preparation of chiral stationary phases using selectors derived from ugi multicomponent condensation reactions and a combinatorial approach.

Combinatorial approaches together with high-throughput screening have been used to develop highly selective stationary phases for chiral recognition. Libraries of potential chiral selectors have been prepared by the Ugi multicomponent condensation reactions and screened for their enantioselectivity using the reciprocal approach involving a chiral stationary phase with immobilized model target compound N-(3,5-dinitrobenzoyl)-alpha-l-leucine. The best candidates were identified from the library of phenyl amides of 2-oxo-azetidineacetic acid derivatives. This screening also enabled specification of the functionalities of the selector desired to achieve the highest level of chiral recognition. The substituents of the phenyl ring adjacent to the chiral center of the selector candidates exhibited the most profound effect on the chiral recognition. The best candidate was then synthesized on a larger scale, resolved into single enantiomers using preparative enantioselective HPLC, and attached to porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) beads via an ester linkage to afford the desired stationary phase. Selectivities alpha as high as 3.2 were found for the separation of a variety of amino acid derivatives.

Efficacy of static magnetic field therapy in chronic pelvic pain: a double-blind pilot study.

OBJECTIVE: The aim of the study was to determine the efficacy of static magnetic field therapy for the treatment of chronic pelvic pain (CPP) by measuring changes in pain relief and disability. STUDY DESIGN: Thirty-two patients with CPP completed 2 weeks and 19 patients completed 4 weeks of randomized double-blind placebo-controlled treatment at a gynecology clinic. Active (500 G) or placebo magnets were applied to abdominal trigger points for 24 hour per day. The McGill Pain Questionnaire, Pain Disability Index, and Clinical Global Impressions Scale were outcome measures. RESULTS: Patients receiving active magnets who completed 4 weeks of double-blind treatment had significantly lower Pain Disability Index (P <.05), Clinical Global Impressions-Severity (P <.05), and Clinical Global Impressions-Improvement (P <.01) scores than those receiving placebo magnets, but were more likely to correctly identify their treatment (P <.05). CONCLUSION: SMF therapy significantly improves disability and may reduce pain when active magnets are worn continuously for 4 weeks in patients with CPP, but blinding efficacy is compromised.

Oral methotrexate for treatment of ectopic pregnancy.

OBJECTIVE: The purpose of this study was to evaluate oral methotrexate tablets in the treatment of ectopic pregnancy. STUDY DESIGN: Patients with a diagnosis of ectopic pregnancy were offered oral methotrexate tablets rather that intramuscular injection. Oral methotrexate was given in 2 divided doses 2 hours apart at a dose of 60 mg/m(2) with standard 2.5 mg methotrexate tablets. Patients were followed up with the use of the same protocol that was used typically for intramuscular methotrexate. RESULTS: Nineteen of 22 patients (86%) were successfully treated. There was no statistical difference between patients who were treated successfully or unsuccessfully, with respect to initial human chorionic gonadotropin titers (P =.55), ectopic size (P =.77), or methotrexate dose (P =.18). Nineteen of 22 patients (86%) had increased pain during treatment. Outside of pain, gastrointestinal side effects were the most common. Thirty-two percent of patients required more than one treatment cycle. CONCLUSION: Oral methotrexate can be used to treat ectopic pregnancy successfully, but there are few advantages to recommend its use over intramuscular methotrexate.

Effect of multivalency on the performance of enantioselective separation media for chiral HPLC prepared by linking multiple selectors to a porous polymer support via aliphatic dendrons.

Chiral stationary phases (CSPs) containing L-proline indananilide chiral selectors attached through a multivalent dendritic linker to monodisperse macroporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) beads have been prepared using two different approaches. The convergent method involves the preparation of ligands in solution and their subsequent attachment to the support. The divergent approach is based on the stepwise "on-bead" formation of the linker using methods that are typical of solid-phase synthesis. While the convergent CSPs feature well-defined ligands, their loading is relatively low. In contrast, the divergent technique affords CSPs with higher loading but with more limited control over precise ligand architecture. Excellent enantioselectivities characterized by separation factors of up to 31 were achieved for the separation of racemic N-(3,5-dinitrobenzoyl)-alpha-amino acid alkyl amides with these new CSPs under normal-phase HPLC conditions.

A new monophasic oral contraceptive containing drospirenone. Effect on premenstrual symptoms.

OBJECTIVE: To determine whether a new monophasic oral contraceptive containing drospirenone/ethinyl estradiol reduces premenstrual symptoms. STUDY DESIGN: In an open-label study measuring intrasubject changes in premenstrual symptoms and comparing effects between women who were new users of oral contraceptives and those who switched from previous contraceptives, ethinyl estradiol (30 micrograms) and drospirenone (3 mg) were administered for 13 menstrual cycles to 326 healthy women aged 18-35 years. Subjects completed the 23-item Women's Health Assessment Questionnaire at baseline and at the end of the sixth cycle. RESULTS: At the end of cycle 6, premenstrual and menstrual symptom scores on the negative affect and water retention scales were reduced significantly relative to baseline, as was increased appetite during the premenstrual and menstrual phases. Similar improvements were seen among new users of hormonal contraceptives and those who switched from previous contraceptives. Impaired concentration scale scores were not significantly reduced from baseline, and assessments of undesired hair changes and feelings of well-being did not change appreciably. CONCLUSION: An oral contraceptive containing drospirenone/ethinyl estradiol may reduce the premenstrual symptoms of negative affect, water retention and increased appetite.