[Neratinib + Valproate] exposure permanently reduces ERBB1 and RAS expression in 4T1 mammary tumors and enhances M1 macrophage infiltration

Abstract

The irreversible ERBB1/2/4 inhibitor neratinib has been shown in vitro to rapidly reduce the expression of ERBB1/2/4 and RAS proteins via autophagic/lysosomal degradation. We have recently demonstrated that neratinib and valproate interact to suppress the growth of 4T1 mammary tumors but had not defined whether the [neratinib + valproate] drug combination, in a mouse, had altered the biology of the 4T1 cells. Exposure of 4T1 mammary tumors to [neratinib + valproate] for three days resulted, two weeks later, in tumors that expressed less ERBB1, K-RAS, N-RAS, indoleamine-pyrrole 2,3-dioxygenase (IDO-1), ornithine decarboxylase (ODC) and had increased Class I MHCA expression. Tumors previously exposed to [neratinib + valproate] grew more slowly than those exposed to vehicle control and contained more CD8+ cells and activated NK cells. M1 but not M2 macrophage infiltration was significantly enhanced by the drug combination. In vitro exposure of 4T1 tumor cells to [neratinib + valproate] variably reduced the expression of histone deacetylases 1-11. In vivo, prior exposure of tumors to [neratinib + valproate] permanently reduced the expression of HDACs 1-3, 6 and 10. Combined knock down of HDACs 1/2/3 or of 3/10 rapidly reduced the expression IDO-1, and ODC and increased the expression of MHCA. H&E staining of normal tissues at animal nadir revealed no obvious cyto-architectural differences between control and drug-treated animals. We conclude that [neratinib + valproate] evolves 4T1 tumors to grow more slowly and to be more sensitive to checkpoint immunotherapy antibodies.

Fingolimod Augments Monomethylfumarate Killing of GBM Cells

“…Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Other reagents and performance of experimental procedures were as described (15,(20)(21)(22)(23)(24). Antibodies used: AIF (5318), BAX (5023), BAK (12105), BAD (9239), BIM (2933), BAK1 (12105), Beclin1 (3495), cathepsin B (31718), CD95 (8023), FADD (2782), eIF2α (5324), P-eIF2α S51 (3398), ULK-1 (8054), P-ULK-1 S757 (14202), P-AMPK S51 (2535), AMPKα (2532), P-ATM S1981 (13050), ATM (2873), ATG5 (12994), mTOR (2983), P-mTOR S2448 (5536), P-mTOR S2481 (2974), ATG13 (13468), MCL-1 (94296), BCL-XL (2764), P-AKT T308 (13038), P-ERK1/2 (5726), P-STAT3 Y705 (9145), P-p65 S536 (3033), p62 (23214), LAMP2 (49067) all from Cell Signaling Technology; P-ULK-1 S317 (3803a) from Abgent; P-ATG13 S318 (19127) from Novus Biologicals.…”

“…Freshly isolated GBM cells and activated microglia directly from the operating room were separated and grown in RPMI supplemented with 2.0% (v/v) fetal calf serum and 10% (v/v) Non-essential amino acids for 6 h, followed by drug exposure and viability assessments made the following day (15,(25)(26)(27). Cells were transfected with siRNA molecules or plasmids as described in prior manuscripts (20)(21)(22)(23)(24). Cells were transfected with a plasmid to express GFP-K-RAS V12 (0.1 µg) using lipofectamine 2000.…”

Palbociclib augments Neratinib killing of tumor cells that is further enhanced by HDAC inhibition

“…Recent studies from our group have demonstrated that the irreversible ERBB1/2/4 inhibitor neratinib can increase autophagosome and autolysosome levels which are responsible for both the down-regulation of RTKs and RAS proteins, as well as causing tumor cell killing. [3][4][5] Thus, we next determined whether the autophagosome-inducing drug palbociclib interacted with the autophagosome-inducing drug neratinib to kill tumor cells. In mammary, colon, NSCLC, sarcoma, pancreatic and renal cancer cells neratinib and palbociclib interacted in an additive to greater than additive fashion to cause tumor cell death after 24h (Figure 2A).…”

“…In tumors, this alteration of biology can be observed up to two weeks after cessation of drug treatments. 5 For example, neratinib and valproate, through down-regulation of HDAC function and expression, alters the protein expression of immunogenic biomarkers. In the present studies we discovered that [neratinib + palbociclib] also could rapidly decrease the expression of PD-L1, PD-L2, IDO-1 and increase the levels of MHCA ( Figure S5).…”

“…Our most recent studies with neratinib, alone or in combination with HDAC inhibitors, have demonstrate a unique biochemical action for the drug. [3][4][5] Neratinib was originally developed as an irreversible inhibitor of ERBB2, and was then shown to inhibit ERBB1 and ERBB4. 6 We discovered that neratinib was both a kinase inhibitor and that it possessed the ability to cause ERBB1/2/3/4 receptor internalization with their subsequent proteolytic degradation.…”