Brentuximab vedotin, an antibody-drug conjugate (ADC), selectively delivers the microtubule-disrupting agent monomethyl auristatin E (MMAE) into CD30-expressing cells. The pharmacokinetics of brentuximab vedotin have been characterized in patients with CD30-positive haematologic malignancies. The primary objective of this phase 1 open label evaluation was to assess the pharmacokinetics of brentuximab vedotin in patients with hepatic or renal impairment.

The aim of this open-label, first-in-setting, randomized phase III trial was to evaluate the efficacy of alisertib, an investigational Aurora A kinase inhibitor, in patients with relapsed/refractory peripheral T-cell lymphoma (PTCL).

Ferroptosis is a form of regulated cell death that can be induced by inhibition of the cystine-glutamate antiporter, system xc-. Among the existing system xc- inhibitors, imidazole ketone erastin (IKE) is a potent, metabolically stable inhibitor of system xc- and inducer of ferroptosis potentially suitable for in vivo applications. We investigated the pharmacokinetic and pharmacodynamic features of IKE in a diffuse large B cell lymphoma (DLBCL) xenograft model and demonstrated that IKE exerted an antitumor effect by inhibiting system xc-, leading to glutathione depletion, lipid peroxidation, and the induction of ferroptosis biomarkers both in vitro and in vivo. Using untargeted lipidomics and qPCR, we identified distinct features of lipid metabolism in IKE-induced ferroptosis. In addition, biodegradable polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticles were employed to aid in IKE delivery and exhibited reduced toxicity compared with free IKE in a DLBCL xenograft model.

While pralatrexate (PDX) has been successfully developed for the treatment of T-cell lymphoma, the mechanistic basis for its T-cell selectivity and acquired resistance remains elusive. In an effort to potentially identify synergistic combinations that might circumnavigate or delay acquired PDX resistance, we generated resistant cells lines over a broad concentration range. PDX-resistant cell lines H9-12 and H9-200 were developed, each exhibiting an IC50 of 35 and over 1000 nM, respectively. These lines were established in vitro from parental H9 cells. Expression analysis of the proteins known to be important determinants of antifolate pharmacology revealed increase expression of dihydrofolate reductase (DHFR) due to gene amplification, and reduced folate carrier1 downregulation, as the putative mechanisms of resistance in H9-12 and H9-200 cells. Cross resistance was only seen with methotrexate but not with romidepsin, azacitidine (AZA), decitabine, gemcitabine, doxorubicin, or bortezomib. Resistance to PDX was reversed by pretreatment with hypomethylating agents in a concentration-dependent fashion. Comparison of gene expression profiles of parental and resistant cell lines confirmed markedly different patterns of gene expression, and identified the dual specificity phosphatase four (DUSP4) as one of the molecular target of PDX activity. Reduced STAT5 phosphorylation following exposure to PDX was observed in the H9 but not in the H9-12 and H9-200 cells. These data suggest that combination with hypomethylating agents could be potent, and that DUSP4 and STAT5 could represent putative biomarkers of PDX activity.

The antiproliferative effect induced by histone deactylase inhibitors (HDACi) is associated with the up-regulated expression of the cyclin-dependent kinase inhibitor p21. Paradoxically, the increased expression of p21 correlates with a reduced cell killing to the drug. The direct targeting of p21 is not feasible. An alternate approach could selectively target factors upstream or downstream of p21 that affect one or more specific aspects of p21 function. HDAC inhibitors appear to activate p21 expression via ataxia telangiectasia mutated (ATM) activity. KU60019, a specific ATM inhibitor, has shown to decrease the p21 protein levels in a concentration dependent manner. We explored the potential synergistic interaction of the ATM inhibitor with romidepsin, given the potential complementary impact around p21. A synergistic cytotoxic effect was observed in all lymphoma cell lines examined when the HDACi was combined with KU60019. The increase in apoptosis correlates with decreased expression of p21 due to the ATM inhibitor. KU60019 decreased expression of the cyclin-dependent kinase inhibitor at the transcriptional level, compromising the ability of HDACi to induce p21 and cell cycle arrest and ultimately facilitating a shift toward the apoptotic phase. Central to the increased apoptosis observed when romidepsin is combined with KU60019 is the reduced expression of p21 and the absence of a G2/M cell cycle arrest that would be exploited by the tumor cells to evade the cytotoxic effect of the HDAC inhibitor. We believe this strategy may offer a promising way to identify rational combinations for HDACi directed therapy, improving their activity in malignant disease.

The peripheral T-cell lymphomas (PTCL) could be considered the prototypical epigenetic disease. As a disease, they are uniquely sensitive to histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors, both alone and in combination, are characterized by a host of mutations in epigenetic genes, and can develop spontaneously in genetically engineered murine models predicated on established recurring mutations in (RHOAG17V) and TET2, an epigenetic gene governing DNA methylation. Given the clinical benefit of HDAC inhibitors (HDACi) and hypomethlyation agents alone and in combination in PTCL, we sought to explore a mechanistic basis for these agents in PTCL. Herein, we reveal profound class synergy between HDAC and DNMT inhibitors in PTCL, and that the combination induces degrees of gene expression that are substantially different and more extensive than that observed for the single agents. A prominent signature of the combination relates to the transcriptional induction of cancer testis antigens and genes involved in the immune response. Interestingly, TBX21 and STAT4, master regulators of TH1 differentiation, were among the genes upregulated by the combination, suggesting the induction of a TH1-like phenotype. Moreover, suppression of genes involved in cholesterol metabolism and the matrisome were also identified. We believe that these data provide a strong rationale for clinical studies, and future combinations leveraging an immunoepigenetic platform.

Multiple myeloma (MM) is the second most common hematologic malignancy. While major advances have been made in the disease, it is still incurable. Although antifolate-based drugs are not commonly used to treat myeloma, new generation analogs with distinct patterns of preclinical and clinical activity may offer an opportunity to identify new classes of potentially active drugs. Pralatrexate (PDX), which was approved for the treatment of relapsed or refractory peripheral T-cell lymphoma in 2009, may be one such drug. Pralatrexate exhibits a potency and pattern of activity distinct from its predecessors like methotrexate (MTX). We sought to understand the activity and mechanisms of resistance of multiple myeloma to these drugs, which could also offer potential strategies for selective use of the drug. We demonstrate that PDX and MTX both induce a significant decrease in cell viability in the low nanomolar range, with PDX exhibiting a more potent effect. We identified a series of myeloma cell lines exhibiting markedly different patterns of sensitivity to the drugs, with some lines frankly resistant, and others exquisitely sensitive. These differences were largely attributed to the basal RFC (Reduced Folate Carrier) mRNA expression levels. RFC mRNA expression correlated directly with rates of drug uptake, with the most sensitive lines exhibiting the most significant intracellular accumulation of pralatrexate. This mechanism explains the widely varying patterns of sensitivity and resistance to pralatrexate in multiple myeloma cell lines. These findings could have implications for this class of drugs and their role in the treatment of multiple myeloma.

Both PD1/PD-L1 and CD47 blockades have demonstrated limited activity in most subtypes of NHL save NK/T-cell lymphoma. The hemotoxicity with anti-CD47 agents in the clinic has been speculated to account for their limitations. Herein we describe a first-in-class and rationally designed bispecific antibody (BsAb), HX009, targeting PD1 and CD47 but with weakened CD47 binding, which selectively hones the BsAb for tumor microenvironment through PD1 interaction, potentially reducing toxicity. In vitro characterization confirmed: (1) Both receptor binding/ligand blockade, with lowered CD47 affinity; (2) functional PD1/CD47 blockades by reporter assays; (3) T-cell activation in Staphylococcal-enterotoxin-B-pretreated PBMC and mixed-lymphocyte-reaction. In vivo modeling demonstrated antitumor activity in Raji-B and Karpass-229-T xenograft lymphomas. In the humanized mouse syngeneic A20 B-lymphoma (huCD47-A20) HuGEMM model, which has quadruple knocked-in hPD1xhPD-L1xhCD47xhSIRPα genes and an intact autologous immune-system, a contribution of effect is demonstrated for each targeted biologic (HX008 targeting PD1 and SIRPα-Fc targeting CD47), which is clearly augmented by the dual targeting with HX009. Lastly, the expression of the immune-checkpoints PD-L1/L2 and CD47 seemed co-regulated among a panel of lymphoma-derived-xenografts, where HX009 maybe more effective in those with upregulated CD47. Our data warrants HX009's further clinical development for treating NHLs.

Histone deacetylase inhibitors (HDACi) induce hyperacetylation of histones by blocking HDAC catalytic sites. Despite regulatory approvals in hematological malignancies, limited solid tumor clinical activity has constrained their potential, arguing for better understanding of mechanisms of action (MOA). Multiple activities of HDACis have been demonstrated, dependent on cell context, beyond the canonical induction of gene expression. Here, using a clinically relevant exposure duration, we established DNA damage as the dominant signature using the NCI-60 cell line database and then focused on the mechanism by which hyperacetylation induces DNA damage. We identified accumulation of DNA-RNA hybrids (R-loops) following romidepsin-induced histone hyperacetylation, with single-stranded DNA (ssDNA) breaks detected by single-cell electrophoresis. Our data suggest that transcription-coupled base excision repair (BER) is involved in resolving ssDNA breaks that, when overwhelmed, evolve to lethal dsDNA breaks. We show that inhibition of BER proteins such as PARP will increase dsDNA breaks in this context. These studies establish accumulation of R-loops as a consequence of romidepsin-mediated histone hyperacetylation. We believe that the insights provided will inform design of more effective combination therapy with HDACis for treatment of solid tumors. IMPLICATIONS: Key HDAC inhibitor mechanisms of action remain unknown; we identify accumulation of DNA-RNA hybrids (R-loops) due to chromatin hyperacetylation that provokes single-stranded DNA damage as a first step toward cell death.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of aggressive non-Hodgkin lymphomas, the majority of which have high relapse rates following standard therapy. Despite use of consolidative stem cell transplant (SCT) following frontline therapy, there remains no consensus on its utility. The double-blind randomized phase 3 ECHELON-2 study (#NCT01777152; clinicaltrials.gov) demonstrated improved progression-free survival (PFS) and overall survival with frontline brentuximab vedotin plus cyclophosphamide, doxorubicin, and prednisone (A+CHP). Herein, we conducted an exploratory subgroups analysis of the impact of consolidative SCT on PFS in patients with previously untreated CD30+ PTCL (ALK- anaplastic large cell lymphoma [ALCL] and non-ALCL) who were in complete response (CR) after frontline treatment with A+CHP or cyclophosphamide, doxorubicin, vincristine, and prednisone. Median PFS follow-up was 47.57 months. The PFS hazard ratio was 0.36, equating to a 64% reduction in the risk of a PFS event in patients who underwent SCT. The median PFS in patients who underwent SCT was not reached, vs 55.66 months in patients who did not undergo SCT. PFS results favored the use of SCT in both ALK- ALCL and non-ALCL subgroups. These data support the consideration of consolidative SCT in patients with CD30+PTCL who achieve CR following treatment with A+CHP.