Antitumor activity of the PI3K δ-sparing inhibitor MEN1611 in PIK3CA mutated, trastuzumab-resistant HER2 + breast cancer.

PURPOSE: Dysregulation of the PI3K pathway is one of the most common events in breast cancer. Here we investigate the activity of the PI3K inhibitor MEN1611 at both molecular and phenotypic levels by dissecting and comparing its profile and efficacy in HER2 + breast cancer models with other PI3K inhibitors. METHODS: Models with different genetic backgrounds were used to investigate the pharmacological profile of MEN1611 against other PI3K inhibitors. In vitro studies evaluated cell viability, PI3K signaling, and cell death upon treatment with MEN1611. In vivo efficacy of the compound was investigated in cell line- and patient-derived xenografts models. RESULTS: Consistent with its biochemical selectivity, MEN1611 demonstrated lower cytotoxic activity in a p110δ-driven cellular model when compared to taselisib, and higher cytotoxic activity in the p110ß-driven cellular model when compared to alpelisib. Moreover, MEN1611 selectively decreased the p110α protein levels in PIK3CA mutated breast cancer cells in a concentration- and proteasome-dependent manner. In vivo, MEN1611 monotherapy showed significant and durable antitumor activity in several trastuzumab-resistant PIK3CA-mutant HER2 + PDX models. The combination of trastuzumab and MEN1611 significantly improved the efficacy compared to single agent treatment. CONCLUSIONS: The profile of MEN1611 and its antitumoral activity suggest an improved profile as compared to pan-inhibitors, which are limited by a less than ideal safety profile, and isoform selective molecules, which may potentially promote development of resistance mechanisms. The compelling antitumor activity in combination with trastuzumab in HER2 + trastuzumab-resistant, PIK3CA mutated breast cancer models is at the basis of the ongoing B-Precise clinical trial (NCT03767335).

CD157: From Myeloid Cell Differentiation Marker to Therapeutic Target in Acute Myeloid Leukemia.

: Human CD157/BST-1 and CD38 are dual receptor-enzymes derived by gene duplication that belong to the ADP ribosyl cyclase gene family. First identified over 30 years ago as Mo5 myeloid differentiation antigen and 10 years later as Bone Marrow Stromal Cell Antigen 1 (BST-1), CD157 proved not to be restricted to the myeloid compartment and to have a diversified functional repertoire ranging from immunity to cancer and metabolism. Despite being a NAD+-metabolizing ectoenzyme anchored to the cell surface through a glycosylphosphatidylinositol moiety, the functional significance of human CD157 as an enzyme remains unclear, while its receptor role emerged from its discovery and has been clearly delineated with the identification of its high affinity binding to fibronectin. The aim of this review is to provide an overview of the immunoregulatory functions of human CD157/BST-1 in physiological and pathological conditions. We then focus on CD157 expression in hematological tumors highlighting its emerging role in the interaction between acute myeloid leukemia and extracellular matrix proteins and its potential utility for monoclonal antibody targeted therapy in this disease.

MEN1309/OBT076, a First-In-Class Antibody-Drug Conjugate Targeting CD205 in Solid Tumors.

CD205 is a type I transmembrane glycoprotein and is a member of the C-type lectin receptor family. Analysis by mass spectrometry revealed that CD205 was robustly expressed and highly prevalent in a variety of solid malignancies from different histotypes. IHC confirmed the increased expression of CD205 in pancreatic, bladder, and triple-negative breast cancer (TNBC) compared with that in the corresponding normal tissues. Using immunofluorescence microscopy, rapid internalization of the CD205 antigen was observed. These results supported the development of MEN1309/OBT076, a fully humanized CD205-targeting mAb conjugated to DM4, a potent maytansinoid derivate, via a cleavable N-succinimidyl-4-(2-pyridyldithio) butanoate linker. MEN1309/OBT076 was characterized in vitro for target binding affinity, mechanism of action, and cytotoxic activity against a panel of cancer cell lines. MEN1309/OBT076 displayed selective and potent cytotoxic effects against tumor cells exhibiting strong and low to moderate CD205 expression. In vivo, MEN1309/OBT076 showed potent antitumor activity resulting in durable responses and complete tumor regressions in many TNBC, pancreatic, and bladder cancer cell line-derived and patient-derived xenograft models, independent of antigen expression levels. Finally, the pharmacokinetics and pharmacodynamic profile of MEN1309/OBT076 was characterized in pancreatic tumor-bearing mice, demonstrating that the serum level of antibody-drug conjugate (ADC) achieved through dosing was consistent with the kinetics of its antitumor activity. Overall, our data demonstrate that MEN1309/OBT076 is a novel and selective ADC with potent activity against CD205-positive tumors. These data supported the clinical development of MEN1309/OBT076, and further evaluation of this ADC is currently ongoing in the first-in-human SHUTTLE clinical trial.

SAHA/Vorinostat induces the expression of the CD137 receptor/ligand system and enhances apoptosis mediated by soluble CD137 receptor in a human breast cancer cell line.

HDAC inhibitors (HDACis) represent a class of anticancer agents including suberoylanilide hydroxamic acid (SAHA, Vorinostat), which has shown a strong antitumor effect, both in vitro and in vivo. Induction of apoptotic genes is an important pathway of SAHA cytotoxic mechanism of action and it has been largely described that SAHA induces sensitization of cell death receptor-resistant breast cancer cells to apoptosis. In this study, we investigated the activation of some apoptotic genes which could be responsible for the in vivo antitumor potency of SAHA in a model of human breast cancer. We found that the apoptotic gene pattern induced by SAHA in the MDA-MB-231 cell line involves the upregulation of some molecules belonging to the TNF superfamily. In particular, we demonstrated that the upregulation of the CD137 receptor/ligand system correlates with a synergistic cytotoxic effect when MDA-MB-231 cells are treated with the combination of SAHA and soluble CD137 receptor. To our knowledge, this is the first study to indicate that this member of the TNF superfamily, CD137, is modulated by SAHA treatment in breast cancer cells, suggesting that the combination of SAHA with this TNF-related receptor could be a new therapeutic approach for the treatment of tumors.

Induction of a less aggressive phenotype in human colon carcinoma HCT116 cells by chronic exposure to HDAC inhibitor SAHA.

Histone deacetylase inhibitors (HDACis) are anticancer molecules that epigenetically modulate cell functions. Chronic exposure of HCT116 colon cancer cells to SAHA has been investigated for a better understanding of resistance mechanisms but, surprisingly, a less aggressive tumor phenotype both in vitro and in vivo was obtained after exposure to increasing concentrations of SAHA. Indeed, HCT116/SAHA cells when injected into nude mice showed a reduced engraftment and growth with respect to HCT116 cells. This difference was not observed inoculating the cells into NOD/SCID mice that, differently from nude mice, lack NK activity, thus suggesting the involvement of the native immune response in impairment of HCT116/SAHA cell growth. In agreement with this result, a growing induction of NKG2D ligand expression, MICA and MICB, that are molecular mediators of NK cell killing, was confirmed in HCT116/SAHA chronically exposed to SAHA. A reduced clonogenic efficiency was also observed in HCT116/SAHA with respect to HCT116 cells. Interestingly, even after chronic exposure to SAHA, HCT116/SAHA cells developed only a moderate resistance to SAHA both in vitro and in vivo and they acquired a collateral sensitivity to anthracyclines. These results are of note and probably rely on the fact that, having simultaneously many different targets, HDACis would require many different mutations to display high resistance index. Moreover, to understand the molecular basis of HCT116/SAHA cell phenotype a gene expression profile of cancer genes was evaluated in HCT116 incubated with SAHA for 24 h and in HCT116/SAHA cells to identify selectively regulated genes.

Antitumor activity of delimotecan against human metastatic melanoma: pharmacokinetics and molecular determinants.

Delimotecan (MEN 4901/T-0128) is a new cytotoxic prodrug constituted by a camptothecin analog (T-2513) bound to carboxymethyl dextran through a triglycine linker. A significant antitumor activity of delimotecan against human metastatic melanoma xenograft model Me15392 is reported. Dacarbazine, the drug approved for the treatment of metastatic melanoma, was ineffective in this melanoma model. Pharmacokinetic studies, together with the expression analysis of mRNA for enzymes involved in delimotecan metabolism, showed that T-2513 and other cytotoxic metabolites of delimotecan (SN 38 and T-0055) are generated in greater quantities in the tumor tissue than in toxicity target tissues, such as liver, thus accounting for the antitumoral activity. Moreover, we demonstrated that human metastatic melanoma cells are able to phagocytose delimotecan and cleave it to release the cytotoxic moieties T-2513 in the tumoral environment. Further flow cytometric analysis showed a higher recruitment of macrophages in xenografted human metastatic melanoma, when compared with other human tumors. Thus, the antitumoral activity of delimotecan exerted on metastatic melanoma is due to several factors: (i) the ability of melanoma cells to phagocytose and metabolise delimotecan; (ii) the accumulation of delimotecan in tumoral mass; (iii) the recruitment of macrophage cells to the melanoma nodule and (iv) the expression in melanoma cells of a pattern of enzymes that converts delimotecan into cytotoxic metabolites. Based on these results, delimotecan might be exploited as a new anticancer agent for the therapy of metastatic melanoma because of its high efficacy and good selectivity, and therefore clinical trials for this indication are warranted.

The synergistic interaction of interferon types I and II leads to marked reduction in severe acute respiratory syndrome-associated coronavirus replication and increase in the expression of mRNAs for interferon-induced proteins.

Interferon (IFN)-alpha, -beta and -gamma have been shown to be only marginally effective against severe acute respiratory syndrome coronavirus (SARS-CoV) replication in Vero cell lines. We investigated the combination of type I IFNs (IFN-alpha or -beta) and IFN-gamma for antiviral activity and found that such combinations synergistically inhibited SARS-CoV replication in Vero cells, using yield reduction assay and the isobologram and combination index methods of Chou and Talalay for evaluation. The highly synergistic anti-SARS-CoV action of type I IFNs and IFN-gamma parallels the marked increase in 2'-5'-oligoadenylate synthetase and p56 mRNAs following exposure in Vero cells to either IFN-alpha or -beta and IFN-gamma compared with the transcriptional levels obtained after stimulation with either IFN alone. These results demonstrate that SARS-CoV, although only moderately sensitive to the antiviral action of the individual types of IFN, is highly sensitive to a combination of type I and II IFNs, which suggests that such combinations may have potential in the treatment of SARS-CoV infections.

Sabarubicin (MEN10755)-induced apoptosis is independent from mtDNA in A2780 human ovarian tumor cells.

BACKGROUND: The role of mitochondrial DNA (mtDNA) in anthracycline-induced apoptosis is controversial. Sabarubicin accumulates in the mitochondria of A2780 human ovarian tumor cells. The effects of this new anthracycline on the structure and the functionality of mtDNA, as well as on the apoptosis of mtDNA-depleted cells have been investigated. MATERIALS AND METHODS: Sabarubicin-induced mtDNA cleavage was detected by Southern blotting and mitochondrial mRNA expression was analyzed by real-time PCR. Apoptosis was studied in mtDNA-depleted (theta0) and parental (theta+) A2780 cells detecting nuclear DNA fragmentation using ELISA and cytofluorimetrically using Annexin V/PI staining. Mitochondrial membrane potential was studied using the cyanine dye JC-1. RESULTS: Sabarubicin induced mtDNA cleavage in the A2780 cells, but this damage did not affect mitochondrial mRNA expression. Apoptosis was induced by sabarubicin in theta0 as well as in theta+ cells. CONCLUSION: The results showed that mtDNA did not influence anthracycline-induced apoptosis in A2780 cells.

Human and murine macrophages mediate activation of MEN 4901/T-0128: a new promising camptothecin analogue-polysaccharide conjugate.

MEN 4901/T-0128 is a new cytotoxic prodrug constituted by the camptothecin analogue T-2513 bound to carboxymethyl dextran through a triglycine linker. MEN 4901/T-0128 was designed to target the active camptothecin at the tumour site. MEN 4901/T-0128 is weakly cytotoxic in vitro and thus T-2513 must be released from the conjugate to become active. Here, we demonstrated that human purified cathepsin B releases T-2513 from MEN 4901/T-0128 at pH values ranging from 3 to 5. pH dependency of this reaction suggests that cleavage of the linker should mainly occur in the lysosomes. As elevated cathepsin B activity has been described in macrophages, human tumour monocytic THP-1 cells differentiated into macrophage-like cells were used to study the cellular mechanisms responsible for MEN 4901/T-0128 antitumour activity. Here, we show that differentiated THP-1 internalizes MEN 4901/T-0128 efficiently in a time-dependent and concentration-dependent manner. After phagocytosis, THP-1 cells can cleave the prodrug and release T-2513 in the media. On the contrary, undifferentiated THP-1 cells or pancreatic ASPC-1 tumour cells, although expressing high levels of cathepsin B, are much less efficient in the release of cytotoxic moieties in the culture media. Moreover, normal murine macrophages, recovered from the peritoneal cavity or from the spleen, when activated (in vitro by 100 ng/ml phorbol 12-myristate-13-acetate and in vivo by 300 microl of 3% w/v thioglycollate solution), were able to release (after incubation with 10 microg/ml MEN 4901/T-0128) cytotoxic moieties in the culture supernatant, in an amount sufficient to kill human carcinoma A2780 cells. Thus, we suggest that tumour-associated macrophages may play a key role in the uptake of MEN 4901/T-0128, cleavage and local release of active moiety T-2513. This mechanism should support a tumour targeting of the cytotoxic moieties, allowing an improved antitumour efficacy/safety ratio for MEN 4901/T-0128.

Sabarubicin- (MEN 10755) and paclitaxel show different kinetics in nuclear factor-kappaB (NF-kB) activation: effect of parthenolide on their cytotoxicity.

BACKGROUND: Several antitumor drugs have been described to induce nuclear factor kappaB (NF-kappaB), but results about its role in regulating apoptotic cell death are quite controversial. In this paper, we studied NF-kappaB induced by the two anticancer agents Sabarubicin (MEN 10755) and paclitaxel (Taxol) and the effects of its pharmacological inhibition. MATERIALS AND METHODS: In the human colon cancer cell line HCT-116, we investigated NF-kappaB activation induced by the two anticancer agents using electrophoretic mobility shift assay (EMSA), while drug-induced cytotoxicity was measured by trypan blue staining. Apoptosis was analyzed using a cell death detection enzyme-linked immunosorbent assay (ELISA) kit, flow cytometry and caspase-3 activation assay. RESULTS: The combination with the NF-kappaB inhibitorparthenolide increased Sabarubicin- but not paclitaxel-induced cell death. EMSA experiments demonstrated that the two antitumor drugs induced NF-kappaB complexes with different kinetics but similar subunit composition. Moreover, Sabarubicin elicited NF-kappaB activation definitely earlier than DNA fragmentation, whereas with paclitaxel the kinetics of the two phenomena were similar.