Ruxolitinib in combination with prednisone and nilotinib exhibit synergistic effects in human cells lines and primary cells from myeloproliferative neoplasms

Ruxolitinib is the front-line non-palliative treatment for myelofibrosis (MF). However, a significant number of patients lose or present suboptimal response, are resistant or have unacceptable toxicity. In an attempt to improve response and avoid the adverse effects of this drug, we evaluated the combination of 17 drugs with ruxolitinib in ex vivo models of peripheral blood mononuclear cells from MF patients and cell lines. We found that the combination ruxolitinib and nilotinib had a synergistic effect against MF cells (ΔEC50 nilotinib, -21.6%). Moreover, the addition of prednisone to combined ruxolitinib/nilotinib improved the synergistic effect in all MF samples studied. We evaluated the molecular mechanisms of combined ruxolitinib/nilotinib/prednisone and observed inhibition of JAK/STAT (STAT5, 69.2+11.8% inhibition) and MAPK (ERK, 29.4+4.5% inhibition) signaling pathways. Furthermore, we found that the triple therapy combination inhibited collagen protein and COL1A1 gene expression in human bone marrow mesenchymal cells. Taken together, we provide evidence that combined ruxolitinib/nilotinib/prednisone is a potential therapy for MF, possibly through the anti-fibrotic effect of nilotinib, the immunomodulatory effect of ruxolitinib and prednisone, and the anti-proliferative effect of ruxolitinib. This combination will be further investigated in a phase Ib/II clinical trial in MF.This study was supported by the Subdireccion General de Investigacion Sanitaria (Instituto de Salud Carlos III, Spain) grants PI13/02387 and PI16/01530, and the CRIS against Cancer foundation grant 2014/0120. M.L. holds a postdoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409).S

Differences in ex-vivo Chemosensitivity to Anthracyclines in First Line Acute Myeloid Leukemia

Induction schedules in acute myeloid leukemia (AML) are based on combinations of cytarabine and anthracyclines. The choice of the anthracycline employed has been widely studied in multiple clinical trials showing similar complete remission rates. Using an ex vivo test we have analyzed if a subset of AML patients may respond differently to cytarabine combined with idarubicin, daunorubicin or mitoxantrone. Bone marrow (BM) samples of 198 AML patients were incubated for 48 hours in 96 well plates, each well containing different drugs or drug combinations at different concentrations. Ex vivo drug sensitivity analysis was made using the PharmaFlow platform maintaining the BM microenvironment. Drug response was evaluated as depletion of AML blast cells in each well after incubation. Annexin V-FITC was used to quantify the ability of the drugs to induce apoptosis, and pharmacological responses were calculated using pharmacokinetic population models. Similar dose-respond graphs were generated for the three anthracyclines, with a slight decrease in EC with idarubicin (p=1.462E-06), whereas the interpatient variability of either drug was large. To identify those cases of selective sensitivity to anthracyclines, potency was compared, in terms of area under the curve. Differences in anthracycline monotherapy potency greater than 30% from 3 pairwise comparisons were identified in 28.3% of samples. Furthermore, different sensitivity was detected in 8.2% of patients comparing combinations of cytarabine and anthracyclines. A third of the patients could benefit from the use of this test in the first line induction therapy selection, although it should be confirmed in a clinical trial specifically designed

A precision medicine test predicts clinical response after idarubicin and cytarabine induction therapy in AML patients

Complete remission (CR) after induction therapy is the first treatment goal in acute myeloid leukemia (AML) patients and has prognostic impact. Our purpose is to determine the correlation between the observed CR/CRi rate after idarubicin (IDA) and cytarabine (CYT) 3 + 7 induction and the leukemic chemosensitivity measured by an ex vivo test of drug activity. Bone marrow samples from adult patients with newly diagnosed AML were included in this study. Whole bone marrow samples were incubated for 48 h in well plates containing IDA, CYT, or their combination. Pharmacological response parameters were estimated using population pharmacodynamic models. Patients attaining a CR/CRi with up to two induction cycles of 3 + 7 were classified as responders and the remaining as resistant. A total of 123 patients fulfilled the inclusion criteria and were evaluable for correlation analyses. The strongest clinical predictors were the area under the curve of the concentration response curves of CYT and IDA. The overall accuracy achieved using MaxSpSe criteria to define positivity was 81%, predicting better responder (93%) than non-responder patients (60%). The ex vivo test provides better yet similar information than cytogenetics, but can be provided before treatment representing a valuable in-time addition. After validation in an external cohort, this novel ex vivo test could be useful to select AML patients for 3 + 7 regimen vs. alternative schedules

A precision medicine test predicts clinical response after idarubicin and cytarabine induction therapy in AML patients

Complete remission (CR) after induction therapy is the first treatment goal in acute myeloid leukemia (AML) patients and has prognostic impact. Our purpose is to determine the correlation between the observed CR/CRi rate after idarubicin (IDA) and cytarabine (CYT) 3 + 7 induction and the leukemic chemosensitivity measured by an ex vivo test of drug activity. Bone marrow samples from adult patients with newly diagnosed AML were included in this study. Whole bone marrow samples were incubated for 48 h in well plates containing IDA, CYT, or their combination. Pharmacological response parameters were estimated using population pharmacodynamic models. Patients attaining a CR/CRi with up to two induction cycles of 3 + 7 were classified as responders and the remaining as resistant. A total of 123 patients fulfilled the inclusion criteria and were evaluable for correlation analyses. The strongest clinical predictors were the area under the curve of the concentration response curves of CYT and IDA. The overall accuracy achieved using MaxSpSe criteria to define positivity was 81%, predicting better responder (93%) than non-responder patients (60%). The ex vivo test provides better yet similar information than cytogenetics, but can be provided before treatment representing a valuable in-time addition. After validation in an external cohort, this novel ex vivo test could be useful to select AML patients for 3 + 7 regimen vs. alternative schedules.This study was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness, Enisa (Empresa Nacional de Innovación, S.A.) and Soprea (Sociedad para la Promoción y Reconversión Económica de Andalucía, S.A.U.)

Ruxolitinib in combination with prednisone and nilotinib exhibit synergistic effects in human cells lines and primary cells from myeloproliferative neoplasms

Ruxolitinib is the front-line non-palliative treatment for myelofibrosis (MF). However, a significant number of patients lose or present suboptimal response, are resistant or have unacceptable toxicity. In an attempt to improve response and avoid the adverse effects of this drug, we evaluated the combination of 17 drugs with ruxolitinib in ex vivo models of peripheral blood mononuclear cells from MF patients and cell lines. We found that the combination ruxolitinib and nilotinib had a synergistic effect against MF cells (ΔEC50 nilotinib, −21.6%). Moreover, the addition of prednisone to combined ruxolitinib/nilotinib improved the synergistic effect in all MF samples studied. We evaluated the molecular mechanisms of combined ruxolitinib/nilotinib/prednisone and observed inhibition of JAK/STAT (STAT5, 69.2+11.8% inhibition) and MAPK (ERK, 29.4+4.5% inhibition) signaling pathways. Furthermore, we found that the triple therapy combination inhibited collagen protein and COL1A1 gene expression in human bone marrow mesenchymal cells. Taken together, we provide evidence that combined ruxolitinib/nilotinib/prednisone is a potential therapy for MF, possibly through the anti-fibrotic effect of nilotinib, the immunomodulatory effect of ruxolitinib and prednisone, and the anti-proliferative effect of ruxolitinib. This combination will be further investigated in a phase Ib/II clinical trial in MF.Subdirección General de Investigación Sanitaria (Instituto de Salud Carlos III, Spain)CRIS against Cancer foundationSpanish Ministry of Economy and CompetitivenessDepto. de Bioquímica y Biología MolecularFac. de FarmaciaTRUEpu

Improving the prediction of acute myeloid leukaemia outcomes by complementing mutational profiling with ex vivo

Refractoriness to induction therapy and relapse after complete remission are the leading causes of death in patients with acute myeloid leukaemia (AML). This study focussed on the prediction of response to standard induction therapy and outcome of patients with AML using a combined strategy of mutational profiling by next-generation sequencing (NGS, n = 190) and ex vivo PharmaFlow testing (n = 74) for the 10 most widely used drugs for AML induction therapy, in a cohort of adult patients uniformly treated according to Spanish PETHEMA guidelines. We identified an adverse mutational profile (EZH2, KMT2A, U2AF1 and/or TP53 mutations) that carries a greater risk of death [hazard ratio (HR): 3 29, P < 0 0001]. A high correlation was found between the ex vivo PharmaFlow results and clinical induction response (69%). Clinical correlation analysis showed that the pattern of multiresistance revealed by ex vivo PharmaFlow identified patients with a high risk of death (HR: 2 58). Patients with mutation status also ran a high risk (HR 4 19), and the risk was increased further in patients with both adverse profiles (HR 4 82). We have developed a new score based on NGS and ex vivo drug testing for AML patients that improves upon current prognostic risk stratification and allows clinicians to tailor treatments to minimise drug resistance.Instituto de Salud Carlos IIICRIS against Cancer foundationSpanish Ministry of Economy and CompetitivenessDepto. de Bioquímica y Biología MolecularFac. de FarmaciaTRUEpu

A precision medicine test predicts clinical response after idarubicin and cytarabine induction therapy in AML patients.

Complete remission (CR) after induction therapy is the first treatment goal in acute myeloid leukemia (AML) patients and has prognostic impact. Our purpose is to determine the correlation between the observed CR/CRi rate after idarubicin (IDA) and cytarabine (CYT) 3 + 7 induction and the leukemic chemosensitivity measured by an ex vivo test of drug activity. Bone marrow samples from adult patients with newly diagnosed AML were included in this study. Whole bone marrow samples were incubated for 48 h in well plates containing IDA, CYT, or their combination. Pharmacological response parameters were estimated using population pharmacodynamic models. Patients attaining a CR/CRi with up to two induction cycles of 3 + 7 were classified as responders and the remaining as resistant. A total of 123 patients fulfilled the inclusion criteria and were evaluable for correlation analyses. The strongest clinical predictors were the area under the curve of the concentration response curves of CYT and IDA. The overall accuracy achieved using MaxSpSe criteria to define positivity was 81%, predicting better responder (93%) than non-responder patients (60%). The ex vivo test provides better yet similar information than cytogenetics, but can be provided before treatment representing a valuable in-time addition. After validation in an external cohort, this novel ex vivo test could be useful to select AML patients for 3 + 7 regimen vs. alternative schedules

Differences in ex-vivo Chemosensitivity to Anthracyclines in First Line Acute Myeloid Leukemia

Induction schedules in acute myeloid leukemia (AML) are based on combinations of cytarabine and anthracyclines. The choice of the anthracycline employed has been widely studied in multiple clinical trials showing similar complete remission rates. Using an ex vivo test we have analyzed if a subset of AML patients may respond differently to cytarabine combined with idarubicin, daunorubicin or mitoxantrone. Bone marrow (BM) samples of 198 AML patients were incubated for 48 hours in 96 well plates, each well containing different drugs or drug combinations at different concentrations. Ex vivo drug sensitivity analysis was made using the PharmaFlow platform maintaining the BM microenvironment. Drug response was evaluated as depletion of AML blast cells in each well after incubation. Annexin V-FITC was used to quantify the ability of the drugs to induce apoptosis, and pharmacological responses were calculated using pharmacokinetic population models. Similar dose-respond graphs were generated for the three anthracyclines, with a slight decrease in EC with idarubicin (p=1.462E-06), whereas the interpatient variability of either drug was large. To identify those cases of selective sensitivity to anthracyclines, potency was compared, in terms of area under the curve. Differences in anthracycline monotherapy potency greater than 30% from 3 pairwise comparisons were identified in 28.3% of samples. Furthermore, different sensitivity was detected in 8.2% of patients comparing combinations of cytarabine and anthracyclines. A third of the patients could benefit from the use of this test in the first line induction therapy selection, although it should be confirmed in a clinical trial specifically designed