The Novel Proteasome Inhibitor Carfilzomib Interacts Synergistically with HDAC Inhibitors in ABC- and GC-DLBCL Cells Both in Vitro and In Vivo through Mechanisms Involving JNK Activation, NF-κB Inactivation, and DNA Damage..
Authors: Dasmahapatra Girija , Lembersky Dmitry , Kramer Lora , Friedberg Jonathan W. , Fisher Richard I , Dent Paul , Grant Steven 
Published: October 2019

Abstract 1688

Poster Board I-714

Recent preclinical studies indicate that proteasome inhibitors (PIs) interact synergistically with histone deacetylase inhibitors (HDACIs) in diverse transformed cell types, including those of hematopoietic origin. Although PIs such as bortezomib display limited single agent activity in diffuse large B-cell lymphoma (DLBCL), regimens combining PIs with histone deacetylase inhibitors (HDACIs) have shown promising activity in multiple myeloma. To improve PI activity in DLBCL, a dual strategy was designed in which the second-generation, irreversible PI carfilzomib (PR-171; CFZ), an agent shown to be active against some bortezomib-resistant cells, was combined with HDACIs in DLBCL cells, including both activated B cell (ABC) and germinal center (GC) sub-types. Co-administration (24-48 hr) of minimally toxic CFZ concentrations (2-8 nM) and modestly toxic concentrations of either vorinostat (vor; 0.75-2.0μM), SNDX-275 (1-2 μM) or SBHA (20-50 μM) resulted in a striking increase in apoptosis (manifested by annexin V positivity, cytochrome c release, and PARP degradation) in various DLBCL cell types, including SUDHL16, SUDHL4, SUDHL6 (GC sub-type) and OCI-LY3, OCI-LY10 (ABC sub-type). Interactions were highly synergistic, as determined by Median Dose Effect analysis, with Combination Index values significantly less than 1.0. Enhanced activity of these regimens was associated with a sharp increase in caspase-3 and -9 activation, mitochondrial damage (loss of σψm), and induction/phosphorylation of the stress kinases JNK and p38 MAPK, accompanied by down-regulation of phospho-AKT. Notably, CFZ blocked vor-induced NF-κB activation in both GC (SUDHL4) and ABC (OCI-LY10) cells. In addition, co-administration of CFZ and HDACIs also induced perturbations in DNA damage/repair processes, including up-regulation of γH2A.X, and acetylation of the DNA repair proteins Ku70 and Ku86. Pharmacologic (e.g. JNK inhibitory peptides) or genetic (e.g., shRNA knockdown or ectopic expression of dominant-negative JNK) interruption of JNK signaling significantly attenuated CFZ/vor- mediated lethality. In contrast, genetic disruption of the AKT pathway (in cells ectopically expressing constitutively active AKT) or pharmacologic inhibition of p38 MAPK (e.g. by SB 203580) did not significantly diminish toxicity. Combined treatment with CFZ and HDAC inhibitors induced G2M arrest in both SUDHL4 and OCI-LY10 cells. While bortezomib-resistant DLBCL cells (SUDHL16-10BR and OCI-LY-40BR) displayed modest cross-resistance to CFZ alone, co-treatment with vor and CFZ dramatically induced apoptosis in resistant cells in association with induction of JNK and DNA damage (γH2A.X) comparable to that observed in parental cells. The CFZ/vor regimen also strikingly induced apoptosis in 3 primary human DLBCL samples. Finally, in vivo administration of CFZ (IV BIW) and vor (IP TIW) to Beige-nude-XID mice (NIH-III) inoculated with SUDHL4 (GC) or OCI-LY10 (ABC) cells substantially suppressed tumor growth compared to single agent treatment. Collectively, these findings indicate that combining HDACIs with CFZ synergistically induces apoptosis in GC- and ABC-DLBCL cells in association with JNK activation, NF-κB inactivation, G2M arrest, and induction of DNA damage. They also suggest that this strategy, which is active against bortezomib-resistant and primary DLBCL cells, and which displays in vivo activity, warrants exploration in refractory DLBCL. Accordingly, plans for a Phase I CFZ/HDACI trial in NHL are underway. Supported by Lymphoma SPORE 1P50 CA130805.

Disclosures

No relevant conflicts of interest to declare.