Pietro Morlacchi

Targeting SH2 domains in breast cancer

Breast cancer is among the most commonly diagnosed cancer types in women worldwide and is the second leading cause of cancer-related disease in the USA. SH2 domains recruit signaling proteins to phosphotyrosine residues on aberrantly activated growth factor and cytokine receptors and contribute to cancer cell cycling, metastasis, angiogenesis and so on. Herein we review phosphopeptide mimetic and small-molecule approaches targeting the SH2 domains of Grb2, Grb7 and STAT3 that inhibit their targets and reduce proliferation in in vitro breast cancer models. Only STAT3 inhibitors have been evaluated in in vivo models and have led to tumor reduction. Taken together, these studies suggest that targeting SH2 domains is an important approach to the treatment of breast cancer.

Targeting the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 6 (STAT6) with Cell-Permeable, Phosphatase-Stable Phosphopeptide Mimics Potently Inhibits Tyr641 Phosphorylation and Transcriptional Activity.

Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure-affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.

Synthesis and in Vitro Evaluation of a Peptidomimetic Inhibitor Targeting the Src Homology 2 (SH2) Domain of STAT6

An improved synthesis of a phosphopeptidomimetic prodrug targeting the Src Homology 2 (SH2) domain of signal transducer and activator of transcription 6 (STAT6) is reported. In our convergent methodology, we employed a phosphotyrosine surrogate active ester harboring pivaloyloxymethyl groups, which efficiently coupled to tert-butylglycinyl proline diarylamide. Biological evaluation of 1 has not been reported. We show that it inhibits STAT6 phosphorylation in intact human bronchial epithelial cells, suggesting potential application in the treatment of asthma.

Abstract PR01: IACS-010759 a novel inhibitor of oxidative phosphorylation advancing into first-in-human studies to exploit metabolic vulnerabilities

Tumor cells normally depend on both glycolysis and oxidative phosphorylation (OXPHOS) to provide the energy and macromolecule building blocks needed to enable continued tumor cell growth. Genetic or epigenetic inactivation of one of these two redundant pathways represents a metabolic vulnerability that should be susceptible to an inhibitor of the other pathway. We have identified multiple contexts where all or a subset of these tumors demonstrate a dependence on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent inhibitor of complex I of oxidative phosphorylation. In isolated mitochondria or permeabilized cells, ATP production or oxygen consumption is inhibited at single digit nM concentrations in the presence of malate/glutamate, but not succinate. More directly, IACS-10759 inhibits the conversion of NADH to NAD+ in an immunoprecipitated complex I assay at low nM concentrations. Importantly, IACS-10759 is orally bioavailable with excellent pharmacokinetics properties in preclinical species, and has an overall profile suitable for clinical development. Our group and others have demonstrated that a variety of tumor types including: AML, plus subsets of lymphoma, breast, melanoma and PDAC are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in multiple cancer types with IACS-10759 led to decreased oxygen consumption rate (OCR). IACS-10759 treatment also led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. In multiple PDX models of primary AML IACS-10759 treatment extends the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and p-AMPK levels. Additionally, tumor growth inhibition or regression was also observed in cell line and PDX xenograft models of lymphoma, triple negative breast, melanoma and PDAC treated with IACS-10759, indicating that subsets of several non-AML indications are also dependent on OXPHOS. Mechanistically, extensive metabolic profiling revealed that the response to IACS-10759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, amino acid biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. As a result of the robust preclinical response in multiple model systems, IACS-10759 has been advanced through IND enabling studies. GLP safety and toxicology have been completed, clinical supplies manufactured, and a Phase I clinical trial in AML will be initiated during the second quarter of 2016. This abstract is also being presented as Poster B35. Citation Format: Philip Jones, M Emilia Di Francesco, Jennifer M. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher A. Bristow, Christopher L. Carroll, Ningping Feng, Jason P. Gay, Mary K. Geck Do, Jennifer M. Greer, Marina Konopleva, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda G. Smith, Sonal Fnu, Jay P. Theroff, Giulio Draetta, Giulio Draetta, Carlo Toniatti, Joseph R. Marszalek. IACS-010759 a novel inhibitor of oxidative phosphorylation advancing into first-in-human studies to exploit metabolic vulnerabilities. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr PR01.

Abstract LB-A15: IACS-010759 is a novel inhibitor of oxidative phosphorylation that selectively targets AML cells by inducing a metabolic catastrophe

Acute myeloid leukemia (AML) is a highly aggressive disease with a high mortality rate that encompasses several genetically and clinically diverse hematological malignancies characterized by clonal expansion of transformed stem/progenitor cells with limited ability to differentiate into mature blood cells. Standard of care for AML has progressed minimally in the past 30 years for relapse/refractory AML, with survival rates of 65 years. Therefore, novel, highly effective therapeutics are needed for this population. Targeting bioenergetic susceptibilities is an exciting area of oncology therapeutics that is potentially applicable in AML. Our group and others have shown that AML blasts depend significantly on mitochondrial oxidative phosphorylation to meet their energy and biomass production demands. Through an extensive medicinal chemistry campaign IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain with excellent PK and a suitable overall profile. In AML cell lines and primary AML blasts treated ex vivo, we observe a robust decrease in proliferation and a concomitant increase in apoptosis with EC50 values of less than 10 nM. Response to IACS-10759 in AML cells was associated with induction of a metabolic catastrophe that negatively impacted the cells9 ability to sustain energy homeostasis, amino acid biosynthesis, and nucleotide production. In a primary AML patient derived xenograft model from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 (QDx5/week) treatment at 10 mg/kg extended the median survival by greater than 2-fold. Inhibition of OXPHOS by IACS-10759 was confirmed in AML cell lines and PDX models by a decrease in oxygen consumption and significant changes in gene and protein expression, non-essential amino acids and nucleotides. Due to the robust response in AML cell lines, primary AML samples ex vivo, and in vivo efficacy in primary AML PDX models, IACS-10759 has been advanced through IND enabling studies with first-in-human studies targeted for the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Maria Alimova, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Sonal, Jay Theroff, Quanyun Xu, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759 is a novel inhibitor of oxidative phosphorylation that selectively targets AML cells by inducing a metabolic catastrophe. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A15.

Small Molecule Targeting of the STAT5/6 Src Homology 2 (SH2) Domains to Inhibit Allergic Airway Disease

Asthma is a chronic inflammatory disease of the lungs and airways and one of the most burdensome of all chronic maladies. Previous studies have established that expression of experimental and human asthma requires the IL-4/IL-13/IL-4 receptor α (IL-4Rα) signaling pathway, which activates the transcription factor STAT6. However, no small molecules targeting this important pathway are currently in clinical development. To this end, using a preclinical asthma model, we sought to develop and test a small-molecule inhibitor of the Src homology 2 domains in mouse and human STAT6. We previously developed multiple peptidomimetic compounds on the basis of blocking the docking site of STAT6 to IL-4Rα and phosphorylation of Tyr641 in STAT6. Here, we expanded the scope of our initial in vitro structure–activity relationship studies to include central and C-terminal analogs of these peptides to develop a lead compound, PM-43I. Conducting initial dose range, toxicity, and pharmacokinetic experiments with PM-43I, we found that it potently inhibits both STAT5- and STAT6-dependent allergic airway disease in mice. Moreover, PM-43I reversed preexisting allergic airway disease in mice with a minimum ED50 of 0.25 μg/kg. Of note, PM-43I was efficiently cleared through the kidneys with no long-term toxicity. We conclude that PM-43I represents the first of a class of small molecules that may be suitable for further clinical development against asthma.

Abstract 4971: IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production to support cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-010759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that AML, plus subsets of glioblastoma, neuroblastoma, lymphoma, melanoma, triple negative breast cancer (TNBC) and pancreatic cancer (PDAC) are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in several cancer types with IACS-010759 led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. Mechanistically, response to IACS-010759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, aspartate biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. Tumor growth inhibition and regression have been observed in molecularly defined subsets of TNBC and PDAC PDX xenograft models treated with IACS-010759, indicating that subsets of these indications are dependent on OXPHOS. Furthermore, treating TNBC or PDAC PDX models post-chemotherapy with IACS-010759 extends progression free survival, consistent with IACS-010759 targeting recently described metabolically adapted residual tumor cells. In orthotopic xenograft models of primary AML cells, daily oral treatment with 1-7.5 mg/kg IACS-010759 extended the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and a gene signature levels. Therefore, these readouts (OCR, aspartate and a nanostring geneset) have been validated for use as exploratory clinical biology of response endpoints. In parallel, completion of preclinical chemistry, manufacturing and control (CMC) as well as GLP safety and tolerability studies with IACS-010759 in multiple species have enabled the selection of a clinical entry dose. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, a Phase I clinical trial in relapsed, refractory AML was initiated in October 2016, with a parallel trial in solid tumors expected to initiate in early 2017. Initial results from the on-going AML trial will be disclosed. Citation Format: Jennifer Molina, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Jason Cross, Naval Daver, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Jing Han, Judy Hirst, Sha Huang, Yongying Jiang, Zhijun Kang, Marina Konopleva, Gang Liu, Helen Ma, Polina Matre, Timothy McAfoos, Funda Meric-Bernstam, Pietro Morlacchi, Florian Muller, Marina Protopopova, Melinda Smith, Sonal Sonal, Yuting Sun, Jay Theroff, Andrea Viale, Quanyun Xu, Carlo Toniatti, Giulio Draetta, Philip Jones, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4971. doi:10.1158/1538-7445.AM2017-4971

Abstract 335: Title: IACS-010759 is a novel clinical candidate that targets AML cells by inducing a metabolic catastrophe through inhibition of oxidative phosphorylation

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production leading to robust cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that a variety of tumor types including: AML, plus subsets of lymphoma, breast, melanoma and PDAC are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in multiple cancer types with IACS-10759 led to decreased oxygen consumption rate (OCR). IACS-10759 treatment also led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. In an orthotopic xenograft model of primary AML cells derived from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 treatment with 3 and 10 mg/kg orally using a 5 on/2 off schedule extended the median survival by greater than 2-fold. Efficacy was paralleled by robust modulation of OCR, aspartate, and p-AMPK levels. Additionally, tumor growth inhibition or regression was also observed in cell line and PDX xenograft models of lymphoma, triple negative breast, melanoma and PDAC treated with IACS-10759, indicating that subsets of several non-AML indications are also dependent on OXPHOS. Mechanistically, extensive metabolic profiling and flux analysis revealed that the response to IACS-10759 was associated with induction of a metabolic imbalance that negatively impacted energy homeostasis, amino acid biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, IACS-10759 has been advanced through IND enabling studies. GLP safety and toxicology have been completed, and we expect to file an IND at the end of 1Q2016 and initiate a Phase I clinical trial in AML during the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Jing Han, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Gera, Jay Theroff, Quanyun Xu, Juliana Velez, Carlo Toniatti, Timothy Heffernan, Giulio Draetta, M. Emilia Di Francesco, Philip Jones, Joseph R. Marszalek. Title: IACS-010759 is a novel clinical candidate that targets AML cells by inducing a metabolic catastrophe through inhibition of oxidative phosphorylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 335.

Extracellular lactate cooperates with limited glucose and glutamine to sustain breast cancer cell survival by providing ATP, NADPH, amino acids, and glutathione

Background Cancer progression occurs upon mutations on regulatory genes that control biological functions including cellular bioenergetics. Such perturbations lead to a metabolic switch that favors aerobic glycolysis and lactate production over oxidative phosphorylation. This process is known as the Warburg effect and results in a lactate-rich tumor microenvironment. The apparently wasteful mechanism has raised the question of why cancer cells switch from the high ATP producing TCA cycle/OXPHOS to glycolysis; and whether lactate serves a biological function in energy metabolism since a high lactate environment correlates with worse patient prognosis in several malignancies including breast cancer. Our goals were to first, determine the fate of carbons from lactate in breast cancer cells; and second, determine the mechanism of lactate metabolism.