Pyridoxal 5′‐phosphate (PLP), the well‐known active form of vitamin B₆, is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in ...response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N‐terminal helix‐turn‐helix motif that binds DNA and an effector‐binding C‐terminal domain homologous to PLP‐dependent enzymes. Although it is known that PLP acts as an anti‐activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA‐binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B₆ vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA‐binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.
Serine hydroxymethyltransferase (SHMT) is a central enzyme in the metabolic reprogramming of cancer cells, providing activated one-carbon units in the serine-glycine one-carbon metabolism. Previous ...studies demonstrated that the cytoplasmic isoform of SHMT (SHMT1) plays a relevant role in lung cancer. SHMT1 is overexpressed in lung cancer patients and NSCLC cell lines. Moreover, SHMT1 is required to maintain DNA integrity. Depletion in lung cancer cell lines causes cell cycle arrest and uracil accumulation and ultimately leads to apoptosis. We found that a pyrazolopyran compound, namely 2.12, preferentially inhibits SHMT1 compared to the mitochondrial counterpart SHMT2. Computational and crystallographic approaches suggest binding at the active site of SHMT1 and a competitive inhibition mechanism. A radio isotopic activity assay shows that inhibition of SHMT by 2.12 also occurs in living cells. Moreover, administration of 2.12 in A549 and H1299 lung cancer cell lines causes apoptosis at LD50 34 μM and rescue experiments underlined selectivity towards SHMT1. These data not only further highlight the relevance of the cytoplasmic isoform SHMT1 in lung cancer but, more importantly, demonstrate that, at least in vitro, it is possible to find selective inhibitors against one specific isoform of SHMT, a key target in metabolic reprogramming of many cancer types.
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 p110β-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).
The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine ...and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP.
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•3-Bromopyruvate completely inhibits SHMT1, alkylating the active site Cys204.•SHMT2, which lacks Cys 204, is only partially inhibited by 3-bromopyruvate.•3-Bromopyruvate binds to SHMT1 active site forming a non-covalent complex.•Introducing a Cys residue in SHMT2 active site yields a complete inhibition.•3-Bromopyruvate is a lead compound for the design of selective SHMT1 inhibitors.
Metabolic reprogramming of tumor cells toward serine catabolism is now recognized as a hallmark of cancer. Serine hydroxymethyltransferase (SHMT), the enzyme providing one-carbon units by converting ...serine and tetrahydrofolate (H4 PteGlu) to glycine and 5,10-CH2 -H4 PteGlu, therefore represents a target of interest in developing new chemotherapeutic drugs. In this study, 13 folate analogues under clinical evaluation or in therapeutic use were in silico screened against SHMT, ultimately identifying four antifolate agents worthy of closer evaluation. The interaction mode of SHMT with these four antifolate drugs (lometrexol, nolatrexed, raltitrexed, and methotrexate) was assessed. The mechanism of SHMT inhibition by the selected antifolate agents was investigated in vitro using the human cytosolic isozyme. The results of this study showed that lometrexol competitively inhibits SHMT with inhibition constant (Ki ) values in the low micromolar. The binding mode of lometrexol to SHMT was further investigated by molecular docking. These results thus provide insights into the mechanism of action of antifolate drugs and constitute the basis for the rational design of novel and more potent inhibitors of SHMT.
SEL24/MEN1703 is a first-in-class, orally available, dual PIM/FLT3 kinase inhibitor currently investigated in patients with Acute Myeloid Leukemia (AML) in the first-in-human study CLI24-001 ...(NCT03008187).
PIM and FLT3 kinases are considered to play an important role in AML and are inhibited by SEL24/MEN1703. Moreover, there is evidence that inhibition of PIM kinases might contribute to overcoming acquired resistance induced by approved FLT3 inhibitors1. In AML, different signal transducers in the FLT3 pathway are substrates of kinases. Therefore, their phosphorylation levels might be modulated by kinase inhibitors and may be exploited as a potential pharmacodynamic biomarker in clinical development. In particular, phosphorylation of S6, 4E-BP1, and STAT5 is regulated by both FLT3 and PIM1/2. Thus, the objective of this investigation was to identify the most promising pharmacodynamic biomarker/s for implementation in the clinical trials of SEL24/MEN1703.
Initially, we assessed the in vitro cytotoxic effect of SEL24/MEN1703 in a panel of 26 AML cell lines harboring different genetic mutations, to identify suitable cell lines for subsequent experiments. In the selected panel of AML cell lines, SEL24/MEN1703 resulted in the inhibition of phosphorylation of S6, 4-EBP1 and STAT5 as measured by immunoblotting. Notably, the reduction in phosphorylated S6 (pS6) in response to SEL24/MEN1703 was particularly evident. Since SEL24/MEN1703 displays a broad cytotoxic activity in AML cell lines, we clustered sensitive and resistant cell lines considering 0.5 μM as the IC50 cut-off value. Then, we investigated the relationship between SEL24/MEN1703 cytotoxic activity in AML cell lines and the inhibition of the above mentioned phosphorylated proteins in a 24-hour cytotoxic assay, showing a correlation between IC50 and the reduction of pS6 (Pearson correlation coefficient: -0.6905, R2= 0.477). To further confirm the in vitro data, SEL24/MEN1703 ability to modulate phosphoproteins was assessed also in xenograft mice bearing MOLM-16 cell line. The phosphorylation status of S6, 4E-BP1 and STAT5 was analyzed by immunoblot in tumor tissues from mice treated at 25 mg/kg of SEL24/MEN1703 at baseline and at 4, 8, and 16 hours after treatment. Results showed that also in vivo, SEL24/MEN1703 administration resulted in a decrease of pS6, with maximum reduction in this parameter observed 4 hours after the administration of the investigational compound.
Based on these results, pS6 was identified as the pharmacodynamic biomarker to be implemented in the CLI24-001 clinical trial. Among different available methods, flow cytometry was selected as the preferred platform to analyze patient samples, because of its ability to provide quantitative assessment of cellular events and pharmacodynamic evaluation in a selected, relevant cell subpopulation, such as the AML blast cells. The assessment of pS6 in the clinical trial is planned both at baseline and at cycle 1 day 14 for whole blood and bone marrow. In addition, pS6 levels will be measured in whole blood at additional time points during treatment cycles. We have implemented the measurement of pS6 in the CLI24-001 trial, and pS6 levels as well as their relationship with the main pharmacokinetic parameters in patients treated with SEL24/MEN703 at 100 and 125 mg will be presented.
1Green A.S. et al., Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia, Sci Adv, 2015
Tomirotti:Menarini Ricerche S.p.A.: Employment. Merlino:Menarini Ricerche S.p.A.: Employment. Fiascarelli:Menarini Ricerche S.p.A.: Employment. Baldini:Menarini Ricerche S.p.A.: Employment. Tagliavini:Menarini Ricerche S.p.A: Employment. Borella:Menarini Ricerche S.p.A.: Employment. Mazan:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Bressan:Menarini Ricerche S.p.A.: Employment. Pellacani:Menarini Ricerche S.p.A.: Employment; Amgen: Equity Ownership. Salerno:Menarini Ricerche S.p.A.: Employment. Binaschi:Menarini Ricerche S.p.A.: Employment. Bellarosa:Menarini Ricerche S.p.A.: Employment.
Purpose
Metastatic hormone receptor-positive (HR+) breast cancer initially responds to serial courses of endocrine therapy, but ultimately becomes refractory. Elacestrant, a new generation ...FDA-approved oral selective estrogen receptor degrader (SERD) and antagonist, has demonstrated efficacy in a subset of women with advanced HR+breast cancer, but there are few patient-derived models to characterize its effect in advanced cancers with diverse treatment histories and acquired mutations.
Methods
We analyzed clinical outcomes with elacestrant, compared with endocrine therapy, among women who had previously been treated with a fulvestrant-containing regimen from the recent phase 3 EMERALD Study. We further modeled sensitivity to elacestrant, compared with the currently approved SERD, fulvestrant in patient-derived xenograft (PDX) models and cultured circulating tumor cells (CTCs).
Results
Analysis of the subset of breast cancer patients enrolled in the EMERALD study who had previously received a fulvestrant-containing regimen indicates that they had better progression-free survival with elacestrant than with standard-of-care endocrine therapy, a finding that was independent estrogen receptor
(ESR1)
gene mutations. We modeled elacestrant responsiveness using patient-derived xenograft (PDX) models and in ex vivo cultured CTCs derived from patients with HR+breast cancer extensively treated with multiple endocrine therapies, including fulvestrant. Both CTCs and PDX models are refractory to fulvestrant but sensitive to elacestrant, independent of mutations in
ESR1
and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (
PIK3CA)
genes.
Conclusion
Elacestrant retains efficacy in breast cancer cells that have acquired resistance to currently available ER targeting therapies. Elacestrant may be an option for patients with HR+/HER2- breast cancer whose disease progressed on fulvestrant in the metastatic setting.
Translational Relevance.
Serial endocrine therapy is the mainstay of management for metastatic HR+breast cancer, but acquisition of drug resistance highlights the need for better therapies. Elacestrant is a recently FDA-approved novel oral selective estrogen receptor degrader (SERD), with demonstrated efficacy in the EMERALD phase 3 clinical trial of refractory HR+breast cancer. Subgroup analysis of the EMERALD clinical trial identifies clinical benefit with elacestrant in patients who had received prior fulvestrant independent of the mutational status of the
ESR1
gene, supporting its potential utility in treating refractory HR+breast cancer. Here, we use pre-clinical models, including ex vivo cultures of circulating tumor cells and patient-derived xenografts, to demonstrate the efficacy of elacestrant in breast cancer cells with acquired resistance to fulvestrant.
Antibody drug conjugates represent an important class of anti-cancer drugs in both solid tumors and hematological cancers. Here, we report preclinical data on the anti-tumor activity of the ...first-in-class antibody drug conjugate MEN1309/OBT076 targeting CD205. The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination and validation experiments on in vivo models. CD205 was first shown frequently expressed in lymphomas, leukemias and multiple myeloma by immunohistochemistry on tissue microarrays. Anti-tumor activity of MEN1309/OBT076 as single agent was then shown across 42 B-cell lymphoma cell lines with a median IC50 of 200 pM and induction of apoptosis in 25/42 (59.5%) of the cases. The activity appeared highly correlated with its target expression. After in vivo validation as the single agent, the antibody drug conjugate synergized with the BCL2 inhibitor venetoclax, and the anti-CD20 monoclonal antibody rituximab. The first-in-class antibody drug targeting CD205, MEN1309/OBT076, demonstrated strong pre-clinical anti-tumor activity in lymphoma, warranting further investigations as a single agent and in combination.
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