Ricolinostat plus lenalidomide, and dexamethasone in relapsed or refractory multiple myeloma: a multicentre phase 1b trial.

BACKGROUND: Histone deacetylase (HDAC) inhibitors are an important new class of therapeutics for treating multiple myeloma. Ricolinostat (ACY-1215) is the first oral selective HDAC6 inhibitor with reduced class I HDAC activity to be studied clinically. Motivated by findings from preclinical studies showing potent synergistic activity with ricolinostat and lenalidomide, our goal was to assess the safety and preliminary activity of the combination of ricolinostat with lenalidomide and dexamethasone in relapsed or refractory multiple myeloma. METHODS: In this multicentre phase 1b trial, we recruited patients aged 18 years or older with previously treated relapsed or refractory multiple myeloma from five cancer centres in the USA. Inclusion criteria included a Karnofsky Performance Status score of at least 70, measureable disease, adequate bone marrow reserve, adequate hepatic function, and a creatinine clearance of at least 50 mL per min. Exclusion criteria included previous exposure to HDAC inhibitors; previous allogeneic stem-cell transplantation; previous autologous stem-cell transplantation within 12 weeks of baseline; active systemic infection; malignancy within the last 5 years; known or suspected HIV, hepatitis B, or hepatitis C infection; a QTc Fridericia of more than 480 ms; and substantial cardiovascular, gastrointestinal, psychiatric, or other medical disorders. We gave escalating doses (from 40-240 mg once daily to 160 mg twice daily) of oral ricolinostat according to a standard 3 + 3 design according to three different regimens on days 1-21 with a conventional 28 day schedule of oral lenalidomide (from 15 mg [in one cohort] to 25 mg [in all other cohorts] once daily) and oral dexamethasone (40 mg weekly). Primary outcomes were dose-limiting toxicities, the maximum tolerated dose of ricolinostat in this combination, and the dose and schedule of ricolinostat recommended for further phase 2 investigation. Secondary outcomes were the pharmacokinetics and pharmacodynamics of ricolinostat in this combination and the preliminary anti-tumour activity of this treatment. The trial is closed to accrual and is registered at ClinicalTrials.gov, number NCT01583283. FINDINGS: Between July 12, 2012, and Aug 20, 2015, we enrolled 38 patients. We observed two dose-limiting toxicities with ricolinostat 160 mg twice daily: one (2%) grade 3 syncope and one (2%) grade 3 myalgia event in different cohorts. A maximum tolerated dose was not reached. We chose ricolinostat 160 mg once daily on days 1-21 of a 28 day cycle as the recommended dose for future phase 2 studies in combination with lenalidomide 25 mg and dexamethasone 40 mg. The most common adverse events were fatigue (grade 1-2 in 14 [37%] patients; grade 3 in seven [18%]) and diarrhoea (grade 1-2 in 15 [39%] patients; grade 3 in two [5%]). Our pharmacodynamic studies showed that at clinically relevant doses, ricolinostat selectively inhibits HDAC6 while retaining a low and tolerable level of class I HDAC inhibition. The pharmacokinetics of ricolinostat and lenalidomide were not affected by co-administration. In a preliminary assessment of antitumour activity, 21 (55% [95% CI 38-71]) of 38 patients had an overall response. INTERPRETATION: The findings from this study provide preliminary evidence that ricolinostat is a safe and well tolerated selective HDAC6 inhibitor, which might partner well with lenalidomide and dexamethasone to enhance their efficacy in relapsed or refractory multiple myeloma. FUNDING: Acetylon Pharmaceuticals.

Hepoxilin A(3) facilitates neutrophilic breach of lipoxygenase-expressing airway epithelial barriers.

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.

Hydrolysis of tumor cell lipids after CTL-mediated death.

Contributions of lipases to CTL function have been debated, including if T cell lipases damage target cells. Expression of the lipase pancreatic lipase-related protein 2 (PLRP2) was previously found in IL-4 cultured lymphocyte cell lines but absent from IL-2 cultured lymphocytes. Here, we evaluated IL-2 and IL-4 induced CTLs for hydrolysis of target cell lipids and killing. Using anti-CD3 redirected lysis of [(3)H]-oleic acid-labeled P815 tumor cells, we detected the release of the radioactive fatty acid (FA). When PLRP2(+/+) and PLRP2(-/-) CTLs were compared, there was more killing by the PLRP2(+/+) CTLs. However, [(3)H]-oleic acid release was similar per dead P815, suggesting that lipid hydrolysis was produced by the dead P815s rather than by PLRP2. The FA release and death were completely dependent on perforin and also occurred when P815s were killed by perforin-containing T cell granule extracts that lacked lipase activity. Death by the cytotoxic granules extracts was unaffected by the addition of lipases. A lipase inhibitor, tetrahydrolipstatin, blocked FA release without affecting CTL-mediated cytotoxicity. Also, CTL-mediated death caused as much FA release as death by disruption of cells by freeze-thawing. The released oleic acid may be sufficient to promote secondary apoptotic responses after CTL-induced trauma.

Regulation of perforin lysis: implications for protein disulfide isomerase proteins.

Perforin, a membrane-permeabilizing protein, is important to T cell cytotoxic action. Perforin has potential to damage the T cell in the endoplasmic reticulum (ER), is sequestered in granules, and later is exocytosed to kill cells. In the ER and after exocytosis, calcium and pH favor perforin activity. We found a novel perforin inhibitor associated with cytotoxic T cell granules and termed it Cytotoxic Regulatory Protein 2 (CxRP2). CxRP2 blocked lysis by granule extracts, recombinant perforin and T cells. Its effects lasted for hours. CxRP2 was calcium stable and refractory to inhibitors of granzyme and cathepsin proteases. Through mass spectrometric analysis of active 50-100 kDa proteins, we identified CxRP2 candidates. Protein disulfide isomerase A3 was the strongest candidate but was unavailable for testing; however, protein disulfide isomerase A1 had CxRP2 activity. Our results indicate that protein disulfide isomerases, in the ER or elsewhere, may protect T cells from their own perforin.

Lipid-dependent cytotoxicity by the lipase PLRP2 and by PLRP2-positive cytotoxic T lymphocytes (CTLs).

IL-4 induces a lipase, pancreatic lipase related protein 2 (PLRP2), in cytotoxic T lymphocytes (CTLs). Because PLRP2 in semen can mediate lipid-dependent toxicity to sperm, we questioned whether CTL-derived PLRP2 could support similar cytotoxicity toward tumor cells. Recombinant PLRP2 was toxic to P815 tumor cells in 48 h when lipid and another protein, colipase, were present. However, PLRP2-positive CTLs (induced with many lots of IL-4) were unable to mediate lipid-dependent cytotoxicity. Notably, CTLs induced with only one lot of IL-4 had lipid-dependent cytotoxicity. The exceptional lot of IL-4 was effective in multiple experiments at inducing lipid-dependent cytotoxicity. The lipid-dependent cytotoxicity it induced was determined to be perforin-independent. CTLs induced with IL-4 that was unable to induce lipid-dependent cytotoxicity had mRNA for PLRP2 but not mRNA for colipase. Therefore, we added exogenous colipase to the CTL assays but still cytotoxicity was unchanged. We conclude (1) that lipid-dependent cytotoxicity, promoted by the lipase PLRP2 and colipase, will kill tumor cells and (2) that more than PLRP2 alone is required for lipid-dependent cytotoxicity mediated by CTLs.

Low dose IL-15 induces snap arming of CD44(low) T lymphocytes in the absence of antigen.

It is widely accepted that naïve T cells require two signals, antigen recognition and co-simulation, to become cytotoxic over the course of 3-5days. However, we observed that freshly isolated murine splenocytes without exposure to antigen become cytotoxic within 24h after culture with IL-15. IL-15 is a cytokine that promotes homeostatic proliferation, maintenance and activation of memory T cells. The induced cytotoxicity, measured by anti-CD3 redirected (51)Cr release, represented the combined activity of T cells regardless of their antigen specificity, and proceeded even when CD44(hi) (memory-associated phenotype) CD8(+) T cells were depleted. Cytotoxic capacity was perforin-dependent and occurred without detectable up-regulation of granzyme B or cell division. After induction, the phenotypic markers for the memory subset and for activation remained unchanged from the expression of resting T cells. Our work suggests that T cells may gain cytotoxic potential earlier than currently thought and even without TCR stimulation.

Pancreatic lipase-related protein 2 (PLRP2) induction by IL-4 in cytotoxic T lymphocytes (CTLs) and reevaluation of the negative effects of its gene ablation on cytotoxicity.

Pancreatic lipase-related protein 2 (PLRP2) is induced by IL-4 in vitro in cytotoxic T lymphocyte (CTL) clones and CTLs from immunized wild-type (WT) PLRP2(+/+) are more cytotoxic than PLRP2(-/-) CTLs, suggesting to previous investigators that the lipase PLRP2 might support CTL functions. Here, we further evaluate PLRP2 in CTLs. We found that PLRP2 was optimally induced in splenocytes by 3.5 x 10(-8) M IL-4 by day 6 after activation and was restricted to CD8(+) T cells. PLRP2 mRNA was detected inconsistently (and at low levels) after activation in the presence of IL-2. Cytotoxicity in 4 h (51)Cr assays of WT CTLs was approximately 3-fold the activity of PLRP2(-/-) CTLs cultured with IL-4 and, with IL-2, was unexpectedly approximately 2 fold the activity of PLRP2(-/-) CTLs. Thus, PLRP2 gene ablation affected short-term (perforin-dependent) cytotoxicity, even under the IL-2 conditions. Other variables failed to account for the reduced cytotoxicity. Granzyme B levels, activation markers, and CD8(+) T cell frequencies were similar for WT vs. PLRP2(-/-) CTLs (with either cytokine). Addition of rPLRP2 to IL-4 induced PLRP2(-/-) CTLs (or to cytotoxic granule extracts) failed to increase lysis, suggesting that the missing mediator is more than released PLRP2. Cytotoxicity of WT and PLRP2(-/-) CTLs was similar in 2-day tumor survival assays with IL-4, which can be mediated by perforin-independent mechanisms. We conclude that extracellular PLRP2 lipase is unable to directly augment the cytotoxicity that was lost by PLRP2 ablation and that after reevaluation, the question of what is PLRP2's role in CD8 T cells is still unanswered.

Sensitization of tumor cells to NK cell-mediated killing by proteasome inhibition.

Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.

Induction of granzyme B and T cell cytotoxic capacity by IL-2 or IL-15 without antigens: multiclonal responses that are extremely lytic if triggered and short-lived after cytokine withdrawal.

The purpose of these studies was to determine the minimal requirements to induce granzyme B, cytotoxic granules and perforin-dependent lytic capacity. To our surprise, both IL-2 and IL-15 induced not only proliferation, but also profound granzyme B and lytic capacity from CD8+ T cells in the absence of antigen or TCR-stimulation. Mouse splenocytes were incubated with mouse r-IL-2 or r-IL-15 for three days, tested by anti-CD3 redirected lysis and examined for intracellular granzyme B and for T cell activation markers. With 10(-8) M IL-2 or IL-15, there was excellent lytic activity at 1:1 effector to target ratios mediated by T cells from wild-type but not from perforin-gene-ablated mice, consistent with multiclonal activation. Lower interleukin concentrations induced less lytic activity. Granzyme B was undetectable on day 0, and greatly elevated on day 3 in CD44hi CD8+ T cells as detected by flow cytometry. Cytokines alone elevated the granzyme B as much as concanavalin A combined with the cytokines. Some ex vivo CD8+ T cells were CD122+, as were the cultured granzyme B+ cells, thus both populations had low-affinity receptors for the interleukins. Only some of the activated cells were proliferating as detected by CFSE labeling. When the cytokines were withdrawn, the cells lost lytic activity within 24 h and then within the next 24 h, died. Our results suggest that high concentrations of either IL-2 or IL-15 will activate the lytic capacity and granzyme B expression of many T cells and that antigen recognition is not required.