Adaptive metabolic reprogramming gives cancer cells a proliferative advantage. Tumour cells extensively use glycolysis to sustain anabolism and produce serine, which not only refuels the one‐carbon ...units necessary for the synthesis of nucleotide precursors and for DNA methylation, but also affects the cellular redox homeostasis. Given its central role in serine metabolism, serine hydroxymethyltransferase (SHMT), a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme, is an attractive target for tumour chemotherapy. In humans, the cytosolic isoform (SHMT1) and the mitochondrial isoform (SHMT2) have distinct cellular roles, but high sequence identity and comparable catalytic properties, which may complicate development of successful therapeutic strategies. Here, we investigated how binding of the cofactor PLP controls the oligomeric state of the human isoforms. The fact that eukaryotic SHMTs are tetrameric proteins while bacterial SHMTs function as dimers may suggest that the quaternary assembly in eukaryotes provides an advantage to fine‐tune SHMT function and differentially regulate intertwined metabolic fluxes, and may provide a tool to address the specificity problem. We determined the crystal structure of SHMT2, and compared it to the apo‐enzyme structure, showing that PLP binding triggers a disorder‐to‐order transition accompanied by a large rigid‐body movement of the two cofactor‐binding domains. Moreover, we demonstrated that SHMT1 exists in solution as a tetramer, both in the absence and presence of PLP, while SHMT2 undergoes a dimer‐to‐tetramer transition upon PLP binding. These findings indicate an unexpected structural difference between the two human SHMT isoforms, which opens new perspectives for understanding their differing behaviours, roles or regulation mechanisms in response to PLP availability in vivo.
MEN1611 is a novel orally bioavailable PI3K inhibitor currently in clinical development for patients with HER2‐positive (HER2+) PI3KCA mutated advanced/metastatic breast cancer (BC) in combination ...with trastuzumab (TZB). In this work, a translational model‐based approach to determine the minimum target exposure of MEN1611 in combination with TZB was applied. First, pharmacokinetic (PK) models for MEN1611 and TZB in mice were developed. Then, in vivo tumor growth inhibition (TGI) data from seven combination studies in mice xenograft models representative of the human HER2+ BC non‐responsive to TZB (alterations of the PI3K/AkT/mTOR pathway) were analyzed using a PK‐pharmacodynamic (PD) TGI model for co‐administration of MEN1611 and TZB. The established PK‐PD relationship was used to quantify the minimum effective MEN1611 concentration, as a function of TZB concentration, needed for tumor eradication in xenograft mice. Finally, a range of minimum effective exposures for MEN1611 were extrapolated to patients with BC, considering the typical steady‐state TZB plasma levels in patients with BC following three alternative regimens (i.v. 4 mg/kg loading dose +2 mg/kg q1w, i.v. 8 mg/kg loading dose +6 mg/kg q3w or s.c. 600 mg q3w). A threshold of about 2000 ng·h/ml for MEN1611 exposure associated with a high likelihood of effective antitumor activity in a large majority of patients was identified for the 3‐weekly and the weekly i.v. schedule for TZB. A slightly lower exposure (i.e., 25% lower) was found for the 3‐weekly s.c. schedule. This important outcome confirmed the adequacy of the therapeutic dose administered in the ongoing phase 1b B‐PRECISE‐01 study in patients with HER2+ PI3KCA mutated advanced/metastatic BC.
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.
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.
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
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.
, 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.
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.
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 (H4PteGlu) to glycine and 5,10‐CH2‐H4PteGlu, 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.
Serine hydroxymethyltransferase (SHMT) has been repeatedly hailed as the missing target for cancer chemotherapy. Here, we identify lometrexol as one of the most potent antifolate inhibitors of human cytosolic SHMT (Kd=2±1 μM) known to date. The results reported represent an initial step toward the development of more potent and effective SHMT inhibitors.
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 p110delta-driven cellular model when compared to taselisib, and higher cytotoxic activity in the p110beta-driven cellular model when compared to alpelisib. Moreover, MEN1611 selectively decreased the p110alpha 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).
Abstract
The PI3K-AKT-mTOR pathway is one of the most dysregulated pathways in human cancers, with PIK3CA, the gene encoding the p110α catalytic isoform of PI3K, representing the most frequently ...mutated gene of this network. In breast tumors, PIK3CA mutations occur in about 20% to 40% of HER2-positive and HER2-negative respectively. The lack of driver oncogenic function, hypothesized for PIK3CA mutations, paralleled by the poor clinical benefit showed by monotherapy with PI3K inhibitors in solid tumors, suggests the use of rational combinations with optimized dosing to improve PI3K inhibitor efficacy. Here we investigate the activity of the PI3K α/β selective and δ-sparing inhibitor MEN1611 in once-daily (QD) at 3.2 mg/kg versus twice-daily (BID) dosing at 1.6 mg/kg, both as a single agent and in combination with trastuzumab, to examine the correlation between drug regimen, target effect, and tumor response. The in vivo studies were investigated with the HER2 amplified, PIK3CA mutated (p.C420R), and trastuzumab-resistant JIMT-1 breast cancer cell line. To compare the effect of QD versus BID MEN1611 treatment, we evaluated the magnitude of PI3K signaling at the tumor site, by measuring the levels of pAKT and pS6 at different time points, at the beginning and the end of treatment. Anti-tumor activity was evaluated in terms of tumor volume inhibition (TVI) and by the modified response evaluation criteria in solid tumors (mRECIST). In monotherapy, although QD dosing produced a slightly longer pathway inhibition (10 vs 8 hours) than the BID, the maximum magnitude achieved was comparable. The analysis of anti-tumor efficacy, showed non-significant differences between the two schedules in terms of tumor volume inhibition (52% versus 45% for the QD and the BID respectively), confirming the PD data. In combo, BID administration was able to keep the pathway efficiently downregulated, counteracting a rebound effect observed at the 24 hour time point in the QD group of animals. Moreover, efficacy evaluation showed significant tumor-regression activity in the BID group of animals compared to the QD (79% versus 43% respectively). Although further studies are needed to evaluate time-dependent drug exposure in tumor and plasma samples, these preliminary findings support the BID clinical schedule of MEN1611 in the B-PRECISE-01 clinical trial (NCT03767335).
Citation Format: Alessio Fiascarelli, Giuseppe Merlino, Stefania Capano, Alessandro Bressan, Mario Bigioni, Andrea Pellacani, Monica Binaschi, Massimiliano Salerno. Dosing frequency/PD/efficacy relationship of MEN1611 in HER2 amplified, PIK3CA mutated, and refractory to Trastuzumab xenograft model of breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1376.
BACKGROUND: Myelofibrosis (MF) is an aggressive myeloproliferative neoplasm. Abnormal growth of hematopoietic stem and progenitor cells in MF is driven by somatic mutations affecting the Janus Kinase ...(JAK) signaling pathway with JAK2 V617F occurring in 50-60% of patients (Cardoso et al, Plos One 2015). The role of the JAK/Signal transducer and activator of transcriptions (STAT) pathway in MF has led to the recent approval of three different JAK2 inhibitors (ruxolitinib, pacritinib, and fedratinib) for MF treatment. However, despite limiting the symptoms of the disease, JAK2 inhibitors do not result in remission due to persistence of JAK2 activation and MF-driving cells (Pandey et al, Blood Cancer J 2022). Thus, there remains a high unmet need for treatment strategies for patients with MF. Tagraxofusp, a first-in-class CD123-directed therapy, is a recombinant fusion protein consisting of human interleukin-3 conjugated to a truncated diphtheria toxin payload approved (US/EU) for the treatment of Blastic Plasmacytoid Dendritic Cell Neoplasm. In MF, CD123 is expressed on malignant cells and bone marrow (BM) accessory cells that support the proliferation of neoplastic cells (Bao et al, Hematol Oncol Stem Cell Ther 2019; Lasho et al, Blood 2014). Based on CD123 expression, tagraxofusp is a potential, novel approach for MF treatment either alone or in combination. Initial results from a two-stage multicenter, phase 2 study (STML-0401-0314; NCT02268253) of single-agent tagraxofusp in pts with relapsed or refractory (R/R) MF demonstrated encouraging clinical efficacy and a manageable safety profile (Yacoub et al. ASH 2021). We aim to further investigate the antitumoral efficacy of tagraxofusp in MF preclinical models and the potential improvement of antitumoral efficacy by combining tagraxofusp with the JAK inhibitors, ruxolitinib, and pacritinib.
METHODS: MF cell lines (SET2, UKE1, HEL: JAK2 V617F; UT7: JAK2 wild type wt) and bone marrow mononuclear cell (BMMC) samples from healthy donors (HD) were characterized for the extracellular expression of CD123 by flow cytometry staining. In vitro cytotoxicity experiments in CD123-positive MF cell lines (SET2, UT7, UKE-1) tested tagraxofusp and ruxolitinib or pacritinib either as single agents or in combination (MTS assay; 72 h). The combination index (CI) was calculated according to the Compusyn-Chou method (Chou TC., Pharmacol Rev, 2006) and Combenefit platform. In vitro co-culture experiments were performed with MF cell lines incubated in the presence of human primary HD-BMMCs. Cell death, as measured by LiveDead/AnnV staining, was monitored by FACS analysis through differential staining of MF cells and BMMCs with Cell Trace Violet dye (CTV). A signaling pathway analysis (JAK/STAT, S6, Mcl1) was performed through immunocapillary electrophoresis on MF cell lines treated with single agents or in combination.
RESULTS: The MF cell lines showed different levels of CD123 expression. Except for the CD123 negative cell line (HEL), CD123 positive cell lines were all sensitive to tagraxofusp independent of CD123 expression intensity. As expected, both ruxolitinib and pacritinib were active in the presence of JAK2 wt pathway. A synergistic drug interaction was observed (mean CI range: 0.59-1.12) in most combinations of tagraxofusp with JAK inhibitors, as shown in a representative CD123+ cell line in Figure 1. When BMMCs, which are a subpopulation of BM Accessory Cells partially expressing CD123, were co-cultured with MF cell lines, the cytotoxic effect induced by tagraxofusp was enhanced, and this improvement was maintained in combination with JAK2 inhibition.
Molecular biomarkers, such as JAK/STAT, S6, and Mcl1, were evaluated in MF cell lines treated with tagraxofusp and JAK inhibitors and supported the MoA of this combination.
CONCLUSIONS: Our studies showed high sensitivity of MF cell lines to tagraxofusp as a single agent, which is expected given phase 2 results demonstrated clinical efficacy of single-agent tagraxofusp in R/R MF (Yacoub et al. ASH 2021). In addition, synergism was observed in combination with JAK inhibitors. The in vitro antitumoral effect was more pronounced in the presence of BM accessory cells, suggesting an extra targeting of CD123-positive BM cells by tagraxofusp in vivo. These results support potential clinical development of TAG in combination with JAK inhibitors for the treatment of MF.
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