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.

Improving NK cell function in multiple myeloma with NKTR-255, a novel polymer-conjugated human IL-15.

Multiple myeloma (MM) is characterized by an immunosuppressive microenvironment that enables tumor development. One of the mechanisms of immune evasion used by MM cells is the inhibition of natural killer (NK) cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal to increase tumor cell recognition, avoid tumor escape and potentially enhancing the effect of other drugs. In this study, we evaluated the ability of the investigational medicine NKTR-255, an IL-15 receptor agonist, to engage the IL-15 pathway and stimulate NK cells against MM cells. We observed that incubation with NKTR-255 was able to tilt the balance toward an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of patients with MM, with increased expression of activating receptors on the surface of treated NK cells. This resulted in an enhanced degranulation, cytokine release, and anti-tumor cytotoxicity when the NK cells were exposed to both MM cell lines and primary MM cells. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared with placebo, weekly injection of the mice with NKTR-255 increased the number of circulating NK cells in peripheral blood and delayed tumor growth. Finally, we observed that combination of NKTR-255 with the anti-CD38 antibody, daratumumab, was effective against MM cells in vitro and in vivo. Taken together, our data suggest a significant impact of NKTR-255 in inducing NK cell function against MM cells with important translational implications.

Biomarker­driven phase Ib clinical trial of OPB­111077 in acute myeloid leukemia.

OPB-111077 is a novel, highly specific oral signal transducer and activator of transcription 3 inhibitor that has exhibited good efficacy against solid and blood cancers, including acute myeloid leukemia (AML), in preclinical models. In the present study, a phase 1b, two-stage, 3+3 dose-escalation clinical trial [dose level (DL)1 of 200 mg/day and DL2 of 250 mg/day on a once daily dose schedule in 28-day cycles] was conducted to assess the maximum tolerated dose (MTD), safety profile and the preliminary antitumor activity of OPB-111077 in patients with high-risk AML. A preliminary preclinical analysis evaluated the anti-proliferative activity of OPB-111077 in 19 patients with AML with a Vivia Biotech ex vivo PharmaFlow precision medicine test. A total of 12 patients were ultimately enrolled in the trial: 5 patients (42%) were treated with DL1, and 7 (58%) were escalated to DL2 of OPB-111077. Dose-limiting toxicities were not observed and the MTD was not reached. In addition, the most frequently reported treatment-emergent adverse events were nausea, vomiting and fatigue. Finally, clinical activity (overall response) was observed in 3 patients (25%). On the whole, the present study demonstrates that OPB-111077 exhibits a good safety and tolerability profile and an acceptable clinical response in patients with high-risk AML. A biomarker-driven design is useful for selecting the study population upfront.

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

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: Similar dose-respond graphs were generated for the three anthracyclines, with a slight decrease in EC50 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. DISCUSSION: 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 novel ex vivo high-throughput assay reveals antiproliferative effects of idelalisib and ibrutinib in chronic lymphocytic leukemia.

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect.

Drug Discovery Testing Compounds in Patient Samples by Automated Flow Cytometry.

Functional ex vivo assays that predict a patient's clinical response to anticancer drugs for guiding cancer treatment have long been a goal, but few have yet proved to be reliable. To address this, we have developed an automated flow cytometry platform for drug screening that evaluates multiple endpoints with a robust data analysis system that can capture the complex mechanisms of action across different compounds. This system, called PharmaFlow, is used to test peripheral blood or bone marrow samples from patients diagnosed with hematological malignancies. Functional assays that use the whole sample, retaining all the microenvironmental components contained in the sample, offer an approach to ex vivo testing that may give results that are clinically relevant. This new approach can help to predict the patients' response to existing treatments or to drugs under development, for hematological malignancies or other tumors. In addition, relevant biomarkers can be identified that determine the patient's sensitivity, resistance, or toxicity to a given treatment. We propose that this approach, which better recapitulates the human microenvironment, constitutes a more predictive assay for personalized medicine and preclinical drug discovery.

Preclinical anti-myeloma activity of EDO-S101, a new bendamustine-derived molecule with added HDACi activity, through potent DNA damage induction and impairment of DNA repair.

BACKGROUND: Despite recent advances in the treatment of multiple myeloma (MM), the prognosis of most patients remains poor, and resistance to traditional and new drugs frequently occurs. EDO-S101 is a novel therapeutic agent conceived as the fusion of a histone deacetylase inhibitor radical to bendamustine, with the aim of potentiating its alkylating activity. METHODS: The efficacy of EDO-S101 was evaluated in vitro, ex vivo and in vivo, alone, and in combination with standard anti-myeloma agents. The underlying mechanisms of action were also evaluated on MM cell lines, patient samples, and different murine models. RESULTS: EDO-S101 displayed potent activity in vitro in MM cell lines (IC50 1.6-4.8 µM) and ex vivo in cells isolated from MM patients, which was higher than that of bendamustine and independent of the p53 status and previous melphalan resistance. This activity was confirmed in vivo, in a CB17-SCID murine plasmacytoma model and in de novo Vk*MYC mice, leading to a significant survival improvement in both models. In addition, EDO-S101 was the only drug with single-agent activity in the multidrug resistant Vk12653 murine model. Attending to its mechanism of action, the molecule showed both, a HDACi effect (demonstrated by α-tubulin and histone hyperacetylation) and a DNA-damaging effect (shown by an increase in γH2AX); the latter being again clearly more potent than that of bendamustine. Using a reporter plasmid integrated into the genome of some MM cell lines, we demonstrate that, apart from inducing a potent DNA damage, EDO-S101 specifically inhibited the double strand break repair by the homologous recombination pathway. Moreover, EDO-S101 treatment reduced the recruitment of repair proteins such as RAD51 to DNA-damage sites identified as γH2AX foci. Finally, EDO-S101 preclinically synergized with bortezomib, both in vitro and in vivo. CONCLUSION: These findings provide rationale for the clinical investigation of EDO-S101 in MM, either as a single agent or in combination with other anti-MM drugs, particularly proteasome inhibitors.

Her-2/neu gene amplification in familial vs sporadic breast cancer. Impact on the behavior of the disease.

We compared the incidence of Her-2/neu amplification in patients with and without a family history of breast cancer and correlated gene status with clinicobiologic and prognostic features in sporadic and familial cases. Of 108 patients, 28.7% had gene amplification. Among 96 cases with family history information available, 28 had an affected first-degree relative. The gene was amplified more frequently in familial than in sporadic cases (13/28 [46%] vs 14/68 [21%]; P = .01). Among familial cases, amplification was associated with adverse clinicobiologic features (poorly differentiated tumors [P = .05], larger tumors [P = .05], more lymph nodes involved [P = .04], and DNA aneuploid [P = .02] and highly proliferative tumors [P = .005]), and the relapse (P = .02) and disease-related death (P = .05) rates were higher than in cases without amplification. Among sporadic cases, amplification was not associated with significantly different disease features, except for a higher incidence of DNA aneuploid tumors (P = .01), percentage of S-phase tumor cells (P = .006), and lower proportion of estrogen (P = .001) and progesterone (P = .002) receptors. Her-2/neu amplification was observed more frequently among patients with a family history of breast cancer, in whom it was associated with adverse clinicobiologic features and a worse clinical outcome.

Pharmacological profiles of acute myeloid leukemia treatments in patient samples by automated flow cytometry: a bridge to individualized medicine.

BACKGROUND: We have evaluated the ex vivo pharmacology of single drugs and drug combinations in malignant cells of bone marrow samples from 125 patients with acute myeloid leukemia using a novel automated flow cytometry-based platform (ExviTech). We have improved previous ex vivo drug testing with 4 innovations: identifying individual leukemic cells, using intact whole blood during the incubation, using an automated platform that escalates reliably data, and performing analyses pharmacodynamic population models. PATIENTS AND METHODS: Samples were sent from 24 hospitals to a central laboratory and incubated for 48 hours in whole blood, after which drug activity was measured in terms of depletion of leukemic cells. RESULTS: The sensitivity of single drugs is assessed for standard efficacy (EMAX) and potency (EC50) variables, ranked as percentiles within the population. The sensitivity of drug-combination treatments is assessed for the synergism achieved in each patient sample. We found a large variability among patient samples in the dose-response curves to a single drug or combination treatment. CONCLUSION: We hypothesize that the use of the individual patient ex vivo pharmacological profiles may help to guide a personalized treatment selection.

Bisursodeoxycholate(ethylenediamine)platinum(II): a new autofluorescent compound. Cytotoxic activity and cell cycle analysis in ovarian and hematological cell lines.

The present paper describes for the first time an intrinsic fluorescent square-planar platinum(II) complex carrying two ursodeoxycholate ligands ([Pt(UDC)2(en)], where UDC(-) = ursodeoxycholate), that emits at room temperature once free in solution. Kinetic studies were carried out in aqueous solution and in the presence of different NaCl concentrations: 4 mM (similar to cytoplasmic concentration) and 150 mM (similar to plasmatic concentration). This novel compound was synthesized from a [PtCl2(en)] complex and shows increased cytotoxic activity against both resting and cycling HeLa cells, with no toxicity for cell lines derived from neoplastic haematopoietic cells.

Impact of BCR/ABL gene expression on the proliferative rate of different subpopulations of haematopoietic cells in chronic myeloid leukaemia.

Despite the effects of BCR ABL on cell proliferation, no study has compared the proliferative rate of different haematopoietic cell compartments from chronic myeloid leukaemia (CML) with those of normal bone marrow (NBM). We comparatively analysed the cell cycle distribution and BCR/ABL expression in different compartments of BM cells from 15 CML and 11 NBM. Overall, our results showed similar proliferative indices in CML patients and NBM. However, CD34+ myeloid precursors from CML patients displayed an increased proportion of S + G2/M-phase cells (P = 0.04), while no significant differences were found between CML and NBM for other BM cell subsets analysed. In BM cells separated by fluorescence-activated cell sorting, decreasing levels of BCR/ABL mRNA were found from CD34+/CD38+ myeloid precursors to myeloblasts; BCR/ABL expression increased afterwards with a peak at the myelocyte/metamyelocyte stage, decreasing in the more mature band/neutrophil compartment. Unexpectedly, BCR/ABL gene expression showed an inverse correlation with the proportion of S + G2/M-phase cells (R = -0.33; P = 0.04). These results suggest that in CML, BCR/ABL expression is associated with an increased proliferation of CD34+ myeloid haematopoietic progenitor cells but not of other more mature myeloid precursors, as confirmed by the observation of an inverse correlation between the amount of BCR/ABL transcripts and the proportion of S + G2/M-phase cells.

Lineage involvement in chronic myeloid leukaemia: comparison between MBCR/ABL and mBCR/ABL cases.

The relationship between different Abelson/breakpoint cluster region (BCR/ABL+) gene rearrangements and the involvement of different haematopoietic cell lineages were investigated in 15 chronic myeloid leukaemia patients. Analysis of purified cell populations confirmed the involvement of the neutrophil (89%), monocytic (89%), eosinophil (88%), erythroid (100%), and CD34(+) cells (100%) in virtually all patients, without differences between minor BCR/ABL+ and major BCR/ABL+ cases; BCR/ABL+ B- and natural killer (NK)-cells were detected in 43% and 31% of cases, respectively, whereas BCR/ABL+ T-cells were rare (7%). All three minor BCR/ABL+ patients showed involvement of both B- and NK-cells, which was infrequent (27%, P = 0.06 and 10%, P = 0.01) among major BCR/ABL+ cases.