Marked Regression Research Articles

Abstract 3403: Drugging the undruggable: Lessons learned from protein phosphatase 2A

Abstract Protein phosphatase 2A (PP2A) is a key tumor suppressor responsible for the dephosphorylation of many oncogenic signaling pathways. The PP2A holoenzyme is comprised of a scaffolding subunit (A), which serves as the structural platform for the catalytic subunit (C) and for an array of regulatory subunits (B) to assemble. Impairment of PP2A is essential for the pathogenesis of many diseases including cancer. In cancer, PP2A is inactivated through a variety of mechanisms including somatic mutation of the Aαsubunit. Our studies show that the most recurrent Aαmutation, P179R, results in an altered protein conformation which prevents the catalytic subunit from binding. Additionally, correcting this mutation, by expressing wild type PP2A Aαin cell lines harboring the P179R mutation, causes a reduction in tumor growth and metastasis. Given its central role in human disease pathogenesis, many strategies have been developed to therapeutically target PP2A.Our lab developed a series of small molecules activators of protein phosphatase 2A. One of our more advanced analogs in this series, DT-061, drives dephosphorylation and degradation of select pathogenic substrates of PP2A such as c-MYC in cellular and in vivo systems. Additionally, we have demonstrated the phosphomimetics of MYC that prevent PP2A mediated dephosphorylation and degradation markedly reduce the anti-tumorigenic activity of this series of PP2A activators further validating the target-substrate specificity of this approach. Specific mutations in the site of drug interaction or overexpression of the DNA tumor virus small T antigen which has been shown to specifically bind to and inactivate PP2A abrogate the in vivo activity of this small molecule series further validating the PP2A specificity of this approach. Importantly, treatment with DT-061 results in tumor growth inhibition in an array of in vivocancer models and marked regressions in combination with MEKi and PARPi.To further define the mechanism of action of this small molecule series, we have used cryo-electron microscopy (cryo-EM) to visualize directly theinteraction between DT-061 and a PP2A heterotrimeric complex. We have identified molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme through the marked prolongation of the kOFF of the native complex. Furthermore, we demonstrate that DT-061 specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate oncogenic targets such as c-MYC in both cellular and in vivo systems. This 3.6 Å structure identifies dynamic molecular interactions between the three distinct PP2A subunits and highlight the inherent mechanisms of PP2A complex assembly and disassembly in both cell free and cellular systems. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for the therapeutic targeting of previously undruggable proteins, and aid in the development of phosphatase-based therapeutics tailored against disease specific phosphor-protein targets. The marriage of multidisciplinary scientific practices has allowed us to present here a previously unrecognized therapeutic strategy of complex stabilization for the activation of endogenous disease combating enzymes. Citation Format: Derek Taylor, Goutham Narla. Drugging the undruggable: Lessons learned from protein phosphatase 2A [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3403.

Abstract CT232: Phase I study of adoptive immunotherapy for advanced MUC1* positive breast cancer with autologous T cells engineered to express a chimeric antigen receptor, huMNC2-CAR44 specific for a cleaved form of MUC1 (MUC1*)

Abstract Background: CAR-T cell therapy targeting CD19 results in marked tumor regression for patients (pts) with B-cell malignancies. It would be ideal to extend the success of CAR-T cell therapy to common epithelial cancers. MUC1* is a post-translationally modified/cleaved form of mucin 1 (MUC1) that is frequently expressed on breast tumors, functions as a growth factor receptor, and is a novel antigen for CAR-T cell therapy. Minerva Biotechnologies developed a CAR (huMNC2-CAR44) that specifically recognizes MUC1* and does not bind to full-length MUC1 or MUC1* negative cells. huMNC2-CAR44 product consists of autologous T cells transduced with a proprietary lentiviral vector encoding humanized MNC2-scFv (MUC1* targeting head), CD8 α; leader, hinge and transmembrane domains, 4-1BB, and CD3ζ domains. Methods: NCT04020575 is a phase I study evaluating the safety of adoptively transferred autologous T cells genetically modified to express huMNC2-CAR44 in pts with metastatic breast cancer (MBC). Key eligibility criteria: MBC of known ER, PR and HER2 status which has MUC1* membrane expression ≥30% by IHC, measurable or evaluable disease, receipt of standard systemic therapies known to confer benefit, age >18, informed consent, adequate organ function, and KPS ≥60%. Key exclusions: active autoimmune disease or uncontrolled infection, contraindication to cyclophosphamide, anticipated survival < 3 months, and/or untreated CNS metastases. After screening, leukapheresis is performed, CD8+ and CD4+ T cells are selected, transduced with huMNC2-CAR44, expanded, and antigen stimulated in vitro. Lymphodepletion with cyclophosphamide and fludarabine is followed 36-96 hours later by infusion of huMNC2-CAR44 CAR-T cells in escalating doses (3.3 x 105 CAR+ T cells/kg - 1 x 107 CAR+ T cells/kg). The primary objective is to identify the maximum tolerated dose of huMNC2-CAR44 T cells by CTCAE v5 and Lee criteria. Secondary objectives include persistence/phenotype of adoptively transferred huMNC2-CAR44 T cells and antitumor activity in pts with measurable disease by RECIST 1.1. Selected exploratory objectives include trafficking of huMNC2-CAR44 T cells to tumor sites, effector function in vivo, association between tumor MUC1* expression and huMNC2-CAR44 T cell response, change in tumor immune microenvironment by multiplex IHC in post-treatment tumor biopsies. Dose escalation or de-escalation is tested using standard 3+3 dose-finding targeting a T cell dose that is associated with a true DLT rate < 33% and > 17%. Once the MTD has been determined, up to 15 more patients will be enrolled in each of 3 expansion cohorts (Luminal, HER2 positive, and TNBC) to explore the anti-tumor activity in these patient populations and to inform future huMNC2-CAR44 T cell trials. Study is open to enrollment. Citation Format: Jennifer M. Specht, David Maloney, Robert Pierce, Monica Dherin, Susan Ra, Quan V. Wu, Cynthia Bamdad. Phase I study of adoptive immunotherapy for advanced MUC1* positive breast cancer with autologous T cells engineered to express a chimeric antigen receptor, huMNC2-CAR44 specific for a cleaved form of MUC1 (MUC1*) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT232.

Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy.

Esophageal cancer is a deadly malignancy with a 5-year survival rate of only 5% to 20%, which has remained unchanged for decades. Esophageal cancer possesses a high frequency of TP53 mutations leading to dysfunctional G1 cell-cycle checkpoint, which likely makes esophageal cancer cells highly reliant upon G2-M checkpoint for adaptation to DNA replication stress and DNA damage after radiation. We aim to explore whether targeting Wee1 kinase to abolish G2-M checkpoint sensitizes esophageal cancer cells to radiotherapy. Cell viability was assessed by cytotoxicity and colony-forming assays, cell-cycle distribution was analyzed by flow cytometry, and mitotic catastrophe was assessed by immunofluorescence staining. Human esophageal cancer xenografts were generated to explore the radiosensitizing effect of AZD1775 in vivo. The IC50 concentrations of AZD1775 on esophageal cancer cell lines were between 300 and 600 nmol/L. AZD1775 (100 nmol/L) as monotherapy did not alter the viability of esophageal cancer cells, but significantly radiosensitized esophageal cancer cells. AZD1775 significantly abrogated radiation-induced G2-M phase arrest and attenuation of p-CDK1-Y15. Moreover, AZD1775 increased radiation-induced mitotic catastrophe, which was accompanied by increased γH2AX levels, and subsequently reduced survival after radiation. Importantly, AZD1775 in combination with radiotherapy resulted in marked tumor regression of esophageal cancer tumor xenografts. Abrogation of G2-M checkpoint by targeting Wee1 kinase with AZD1775 sensitizes esophageal cancer cells to radiotherapy in vitro and in mouse xenografts. Our findings suggest that inhibition of Wee1 by AZD1775 is an effective strategy for radiosensitization in esophageal cancer and warrants clinical testing.

Myxoma Virus-Loaded Mesenchymal Stem Cells in Experimental Oncolytic Therapy of Murine Pulmonary Melanoma

Oncolytic viruses can target neoplasms, triggering oncolytic and immune effects. Their delivery to melanoma lesions remains challenging. Bone-marrow-derived mesenchymal stem cells (MSCs) were shown to be permissive for oncolytic myxoma virus (MYXV), allowing its transfer to melanoma cells, leading to their killing. Involvement of progeny virus was demonstrated in the transfer from MSCs to co-cultured melanoma cells. The inhibitory effect of virus on melanoma foci formation in murine lungs was revealed using melanoma cells previously co-cultured with MYXV-infected MSCs. Virus accumulation and persistence in lungs of lesion-bearing mice were shown following intravenous administration of MSC-shielded MYXV construct encoding luciferase. Therapy of experimentally induced lung melanoma in mice with interleukin (IL)-15-carrying MYXV construct delivered by MSCs led to marked regression of lesions and could increase survival. Elevated natural killer (NK) cell percentages in blood indicated robust innate responses against unshielded virus only. Lung infiltration by NK cells was followed by inflow of CD8+ T lymphocytes into melanoma lesions. Elevated expression of genes involved in adaptive immune response following oncolytic treatment was confirmed using RT-qPCR. No adverse pathological effects related to MSC-mediated oncolytic therapy with MYXV were observed. MSCs allow for safe and efficient ferrying of therapeutic MYXV to pulmonary melanoma foci triggering immune effects.

Chemogenomic profiling of breast cancer patient-derived xenografts reveals targetable vulnerabilities for difficult-to-treat tumors

Subsets of breast tumors present major clinical challenges, including triple-negative, metastatic/recurrent disease and rare histologies. Here, we developed 37 patient-derived xenografts (PDX) from these difficult-to-treat cancers to interrogate their molecular composition and functional biology. Whole-genome and transcriptome sequencing and reverse-phase protein arrays revealed that PDXs conserve the molecular landscape of their corresponding patient tumors. Metastatic potential varied between PDXs, where low-penetrance lung micrometastases were most common, though a subset of models displayed high rates of dissemination in organotropic or diffuse patterns consistent with what was observed clinically. Chemosensitivity profiling was performed in vivo with standard-of-care agents, where multi-drug chemoresistance was retained upon xenotransplantation. Consolidating chemogenomic data identified actionable features in the majority of PDXs, and marked regressions were observed in a subset that was evaluated in vivo. Together, this clinically-annotated PDX library with comprehensive molecular and phenotypic profiling serves as a resource for preclinical studies on difficult-to-treat breast tumors.

Disappearing Villages in Poland – Selected Socioeconomic Processes and Spatial Phenomena

Abstract The work was to diagnose rural settlements that have been disappearing gradually since the end of World War II as a result of a marked process of depopulation. The authors focused on the disappearance phenomenon itself, seeking to identify and assess selected socioeconomic and spatial processes ongoing in the areas concerned. Through analysis of the set of 41,466 statistical localities in rural areas of Poland, it was found that 62% of these villages reported a decline in the number of inhabitants over the 1950–2011 period. Throughout the post-War period, it is small villages of fewer than 100 people that have experienced the most marked demographic regress. The smallest settlements of all – with 50 people or less – have suffered the most-marked loss of population, at around 28% on average. To be noted, among the factors behind disappearances of villages are limited attractiveness arising out of low standards of living, natural conditions unsuited to farming, limited incomes earned from agricultural activity and poor outfitting in social and technical infrastructure.

Vav1 and mutant K-Ras synergize in the early development of pancreatic ductal adenocarcinoma in mice.

To explore the contribution of Vav1, a hematopoietic signal transducer, to pancreatic ductal adenocarcinoma (PDAC) development, we generated transgenic mouse lines expressing, Vav1, K-RasG12D, or both K-RasG12D and Vav1 in pancreatic acinar cells. Co-expression of Vav1 and K-RasG12D synergistically enhanced acinar-to-ductal metaplasia (ADM) formation, far exceeding the number of lesions developed in K-RasG12D mice. Mice expressing only Vav1 did not develop ADM. Moreover, the incidence of PDAC in K-RasG12D/Vav1 was significantly higher than in K-RasG12D mice. Discontinuing Vav1 expression in K-RasG12D/Vav1 mice elicited a marked regression of malignant lesions in the pancreas, demonstrating Vav1 is required for generation and maintenance of ADM. Rac1-GTP levels in the K-RasG12D/Vav1 mice pancreas clearly demonstrated an increase in Rac1 activity. Treatment of K-RasG12D and K-RasG12D/Vav1 mice with azathioprine, an immune-suppressor drug which inhibits Vav1's activity as a GDP/GTP exchange factor, dramatically reduced the number of malignant lesions. These results suggest that Vav1 plays a role in the development of PDAC when co-expressed with K-RasG12D via its activity as a GEF for Rac1GTPase.

Targeting Doublecortin‐like Kinase 1 (DCLK1) in Colon Cancer: An In‐silico Approach

Cancer stem cells (CSCs) are proposed to be a contributing factor in colorectal cancer (CRC) recurrence and chemo‐resistance, as a result of increased expression of ABC transporters and their innate ability to self‐renew and enter a quiescent state. Current standard therapies are ineffective at eliminating these CSC populations, hence there is a need to identify novel agents to effectively inhibit their growth. Multiple surface markers have been identified for CRC‐CSCs, though few have shown promise as a clinical target. Recently, doublecortin‐like kinase 1 (DCLK1) was established as a CSC marker in CRC and pancreatic cancers. The N‐terminal doublecortin domains(DCX) bind microtubules and regulate their polymerization, while the C‐terminal serine/threonine‐protein kinase domain is involved in cellular signaling. DCLK1 is a marker of Tuft cells in normal intestinal crypts, but in cancer is found to be highly expressed in CSC populations that play a critical role in tumor progression, based on lineage tracing experiments. Specific ablation of DCLK1+ stem cells causes marked regression of polyps without apparent damage to the normal intestine, distinguishing DCLK1 as a CSC marker. Recently, marmelin, a phytochemical from Aegle marmelos, has been shown to target DCLK1 and reduce CRC tumor growth in vivo. Hence, DCLK1 represents an attractive target for CSC‐targeted drug discovery. The present study is aimed to identify small molecule inhibitors of DCLK by using in silico molecular docking‐based screening. For this, we tested a library of FDA‐approved drugs and kinase inhibitors using Autodock Vina software. Compounds were assessed on their ability to bind within the ligand binding site of the C‐terminal kinase domain. The compounds are ranked‐based on their binding energies and hydrogen bonding patterns.Support or Funding InformationThe Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationship in this project.

A Rare Case of Primary CNS Germinoma in the Pineal Region with Long-standing Hearing Loss

Germinomas in the brain are rare neoplasms which make up less than 1% of all intracranial tumors. Their clinical presentation is usually related to the increased intracranial pressure and/or hydrocephalus. This report describes a case who presented to our hospital for headaches and visual changes, including Parinaud's syndrome, that had started recently. The patient also had been complaining of chronic bilateral hearing loss for several years which seemed not properly evaluated previously. MRI brain showed a markedly enhancing lesion in the pineal region. CSF and serum tumor markers including B-human chorionic gonadotropin (B-HCG) and alfa-fetoprotein (AFP) were normal. Lumbar puncture with cytology was negative for malignancy. Biopsy was taken from the mass and sent to the pathology lab for testing. Histopathological examination showed changes consistent with germinoma and the immunohistochemical staining including that for placental alkaline phosphatase (PLAP) confirmed the diagnosis. There was no evidence of metastasis and the patient was treated with radiation-alone therapy that resulted in marked regression of the mass and significant clinical improvement except for the issue of hearing loss which had persisted, as expected, given its long-standing existance. In addition to highlighting the diagnostic evaluation and treatment approach for such rare tumors, this case suggests considering pineal germinomas among the differential diagnosis when dealing with hearing loss which needs careful evaluation and should never be underestimated.

Phase I study of pasotuxizumab (AMG 212/BAY 2010112), a PSMA-targeting BiTE (Bispecific T-cell Engager) immune therapy for metastatic castration-resistant prostate cancer (mCRPC).

124 Background: mCRPC has a poor prognosis and immunotherapies are largely ineffective. PSMA is a promising therapeutic target in mCRPC. Pasotuxizumab is a PSMA x CD3 BiTE immune therapy that mediates killing of tumor cells by T cells. Methods: NCT01723475 was a first-in-human, multicenter, dose-escalation study in patients (pts) with mCRPC refractory to standard therapy. Pts received continuous IV infusion of pasotuxizumab in cohorts of 3–4 pts. Dose-escalation followed a continuous reassessment methodology. The primary objective was to determine safety and maximum tolerated dose (MTD); secondary objectives included pharmacokinetics, biomarkers, and tumor response. Results: 16 pts were enrolled into 5 dosing cohorts (5 µg/d, n=3; 10 µg/d, n=4; 20 µg/d, n=3; 40 µg/d, n=4; 80 µg/d, n=2). All pts had ≥1 AE of any grade; most common were fever (94%), chills (69%), and fatigue (50%). 13 pts (81%) had ≥1 AE of grade ≥3; most common were decreased lymphocytes and infections (both 44%). No grade 5 AE occurred. A serious drug-related AE was reported for 1 pt (fatigue, 20 µg/d). No antidrug antibodies were observed. Recruitment was stopped before MTD was reached to allow initiation of a new study sponsored by Amgen. Antitumor activity as indicated by PSA serum level decline was dose dependent, with a mean best PSA change per dosing cohort vs baseline of +0.74% (5 µg/d), −17.9% (10 µg/d), −37.4% (20 µg/d), −42.5% (40 µg/d) and −54.9% (80 µg/d). PSA decreases ≥50% occurred in 3 pts (n=1 each in 20 µg/d, 40 µg/d, 80 µg/d cohorts). One long-term PSA responder was treated for 14 months (40 µg/d) and one for 19.4 months (80 µg/d). The latter pt showed a complete regression of soft-tissue metastases and marked regression of bone metastases by PSMA-PET/CT, >90% reduction in PSA and alkaline phosphatase, and a significant and durable improvement in disease related symptoms. Conclusions: Pasotuxizumab had an acceptable safety profile and dose-dependent clinical activity in mCRPC pts. There were two long term responders in the dose escalation. This is the first clinical study showing that a BiTE immune therapy can be efficacious in solid tumors. Clinical trial information: NCT01723475.

Adding Indoximod to Hypofractionated Radiotherapy with Anti-PD-1 Checkpoint Blockade Enhances Early NK and CD8+ T-Cell-Dependent Tumor Activity.

There is growing interest in combinations of immunogenic radiotherapy (RT) and immune checkpoint blockade, but clinical responses are still limited. Therefore, we tested the triple therapy with an inhibitor of the indoleamine 2,3-dioxygenase pathway, which like immune checkpoints, downregulates the antitumor immune response. Triple treatment with hypofractionated RT (hRT) + anti-PD-1 antibody (αPD1) + indoximod was compared with the respective mono- and dual therapies in two syngeneic mouse models. The tumors did not regress following treatment with hRT + αPD1. The αPD1/indoximod combination was not effective at all. In contrast, triple treatment induced rapid, marked tumor regression, even in mice with a large tumor. The effects strongly depended on CD8+ T cells and partly on natural killer (NK) cells. Numbers and functionality of tumor-specific CD8+ T cells and NK cells were increased, particularly early during treatment. However, after 2.5-3 weeks, all large tumors relapsed, which was accompanied by increased apoptosis of tumor-infiltrating lymphocytes associated with a non-reprogrammable state of exhaustion, terminal differentiation, and increased activation-induced cell death, which could not be prevented by indoximod in these aggressive tumor models. Some mice with a smaller tumor were cured. Reirradiation during late regression (day 12), but not after relapse, cured almost all mice with a large B16-CD133 tumor, and strongly delayed relapse in the less immunogenic 4T1 model, depending on CD8+ T cells. Our findings may serve as a rationale for the clinical evaluation of this triple-combination therapy in patients with solitary or oligometastatic tumors in the neoadjuvant or the definitive setting.

Wsie zanikające – identyfikacja i analiza wybranych cech społeczno-ekonomicznych = Disappearing villages – identification and analysis of selected socioeconomic features

Contemporary rural settlement in Poland is first and foremost modified by demographic processes, of which decisive ones are a migratory influx of new inhabitants into rural areas in the immediate vicinity of large cities, as well as population outflow from most rural areas that are located traditionally and above all peripherally. These two directions to migration generate mostly opposing trends where the development of rural areas is concerned. Inflows of new inhabitants into suburbs and satellite villages close to cities ensure social, economic and infrastructural development or ”progress”, and sometimes even – paradoxically – a change of status from rural to urban. On the other hand, migratory outflows from most other rural areas give rise to a process of demographic degradation whose extreme manifestation may be the actual disappearance of a rural settlement. The fundamental unit of measurement and study here was the so-called ”statistical locality”, defined by GUS as the group of places taking on the name of the leading locality and usually comprising a village plus smaller settlements adjacent to it. The research encompassed a total of 41,466 such localities existing uninterruptedly between 1950 and 2011. The analysis further encompassed six time intervals, of which each ended in a year during which a Census was carried out (i.e. 1950, 1960, 1970, 1978, 1988, 2002 and 2011). For each inter-Censal period, attention was paid to the magnitude and direction of demographic change taking place in all of the rural localities. In this work, the key focus is on the “uniformly regressing villages”, which is to say those losing more than 5% of their inhabitants in the 1950‒2011 period, but also experiencing population declines in every one of the six inter-Censal periods. This is taken to be adequate justification for use of the more-loaded term “disappearing”. Through analysis of the statistical localities in rural areas it was found that some 25,711 of all villages reported a decline in the number of inhabitants over the 1950‒2011 period. However, the steady loss of people indicated by population declines over each of the six studied time intervals was an issue for 2956 of the villages mainly located in central and eastern parts of Poland. These are areas long regarded as only poorly-developed economically, including parts of the east that are clearly categorisable as “peripheral”. To be noted among the factors behind disappearances of villages are limited attractiveness arising out of low standards of living, natural conditions unsuited to farming, limited incomes earned from agricultural activity and poor outfitting in social and technical infrastructure. Throughout the post-War period it is small villages of fewer than 100 people that have experienced the most marked demographic regress. The smallest settlements of all – with 50 people or less – have experienced the most major loss of population, at around 28% on average. The analyses reported here thus support the notion that the scale and village, the less-serious the unfavourable population processes. However, the disappearing villages are home to a number of overlapping problems with development. Inhabitants either lack opportunities to meet their daily needs, or at best have limited chances to do so. And this starts with physiological needs, though also of course ranges through the social, emotional and economic, and ends with lack of access to public goods and services. The decline of rural areas thus features a so-called triggering factor, with this then unleashing a sequence of positive-feedback processes. A most typical trigger would be lack of job opportunities, and this exerts a selective effect in relation to who leaves the countryside. A next consequence of that is a decline in the purchasing power of rural households, a further worsening of living conditions, and inefficient use of infrastructure.

Abstract C069: The identification of MRTX849, a novel KRASG12C inhibitor under clinical investigation, provides insight toward therapeutic susceptibility of KRAS mutant cancers

Abstract The ability to effectively target mutated KRAS has remained elusive despite decades of research. MRTX849 was identified via structure-based drug design as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties. MRTX849 is presently under evaluation in clinical trials and its discovery and evaluation is disclosed here for the first time. MRTX849 demonstrated selective modification of the mutant cysteine residue at amino acid residue 12 in GDP-bound KRASG12C and inhibited KRAS-dependent signaling in vitro and in vivo. In multiple KRASG12C positive cell lines in vitro, MRTX849 treatment demonstrated covalent modification of mutant KRAS and/or inhibition of active KRAS at concentrations as low as 2 nM with near maximal inhibition observed at 15 nM. In vivo, clear evidence of dose-dependent modification of KRASG12C and inhibition of KRAS-dependent signal transduction was observed in multiple KRASG12C mutant tumor models. MRTX849 demonstrated marked tumor regression in 17 out of 26 (65%) of KRASG12C-positive cell line- and patient-derived xenograft (PDX) models but not in tumor models without KRASG12C mutations. While MRTX849 commonly elicited tumor regression, a subset of models was less sensitive to treatment or exhibited tumor stasis after an initial response phase. Comprehensive pharmacodynamic and pharmacogenomic profiling in these models identified mechanisms implicated in limiting anti-tumor response in refractory models including extrinsic factors impacting KRAS nucleotide cycling and/or signaling pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included upstream activation of ERBB family receptor tyrosine kinases (RTKs) as well as genetic dysregulation of cell cycle transition genes. Based on these observations, combination strategies designed to co-target signaling feedback and bypass pathways were evaluated in multiple tumor models. Afatinib and the SHP2 inhibitor, RMC-4550, in combination with MRTX849 validated these therapeutic hypotheses and the combinations demonstrated marked tumor regression in several tumor models, including models that were refractory to either single agent. MRTX849 in combination with the CDK4/6 inhibitor palbociclib also demonstrated a marked antitumor response; notably in tumor models harboring CDKN2A deletion or other genetic alterations implicated in cell cycle dysregulation. Together, these data indicate the therapeutic susceptibility and broad dependence of KRAS G12C mutation-positive tumors on KRAS for tumor cell growth and survival and provide insight toward the molecular basis of response to single agent and combinatorial therapies. Citation Format: James G Christensen, Jay B Fell, Jill Hallin, Brian Baer, Lars engstrom, James Blake, David Briere, Josh Ballard, Michael Burkhard, John Fischer, Guy Vigers, Ruth Aranda, Vickie Bowcut, Andrew Calinisan, Lauren Hargis, Niranjan Sudhakar, Matt Marx, Peter Olson. The identification of MRTX849, a novel KRASG12C inhibitor under clinical investigation, provides insight toward therapeutic susceptibility of KRAS mutant cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C069. doi:10.1158/1535-7163.TARG-19-C069

Abstract B073: Development of combination therapies to potentiate the impact of KRAS-G12C inhibitors in lung cancer

Abstract Oncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. We have previously shown that NSCLC cells harboring KRAS mutations are selectively sensitive to inhibition of MEK and IGF1R signaling. Using a whole-genome shRNA screen, we shown that this effect is markedly enhanced by simultaneous inhibition of mTOR, while maintaining selectivity for the KRAS mutant genotype. Mechanistic investigations demonstrated that IGF1R inhibitors block the reactivation of the PI3K pathway induced by mTOR inhibition, resulting in a robust suppression of PI3K and mTORC1 signalling. Addition of a MEK inhibitor to the combination results in simultaneous inhibition of the main nodes downstream of RAS. Notably, the inhibition of these signalling pathways is stronger in KRAS-mutant cells than in their wild-type counterparts. Combining MEK, IGF1R and mTOR inhibitors produces a profound and durable suppression of cell proliferation and a stronger induction of apoptosis, resulting in marked tumor regression in three different Kras-driven lung cancer mouse models. In order to achieve strong downstream pathway inhibition even more specifically in KRAS-mutant cells and reduce potential unwanted toxicities, we used KRAS-G12C mutant inhibitors. We show that the triple combination of mTOR, IGF1R and KRAS-G12C inhibitors causes a profound and durable inhibition of the main pathways downstream RAS and greatly improves the effectiveness of ARS-1620 on KRAS-G12C mutant lung cancer cells in vitro and in mouse models. This provides a clear rationale for the design of combination treatments to enhance and extend the response to KRAS-G12C inhibitors. Citation Format: Miriam Molina-Arcas, Christopher Moore, Sareena Rana, Febe Van Maldegem, Edurne Mugarza, Pablo Romero-Clavijo, Stuart Horswell, David Hancock, Julian Downward. Development of combination therapies to potentiate the impact of KRAS-G12C inhibitors in lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B073. doi:10.1158/1535-7163.TARG-19-B073

Rational combination with PDK1 inhibition overcomes cetuximab resistance in head and neck squamous cell carcinoma.

Cetuximab, an EGFR-blocking antibody, is currently approved for treatment of metastatic head and neck squamous cell carcinoma (HNSCC), but its response rate is limited. In addition to blocking EGFR-stimulated cell signaling, cetuximab can induce endocytosis of ASCT2, a glutamine transporter associated with EGFR in a complex, leading to glutathione biosynthesis inhibition and cellular sensitization to ROS. Pyruvate dehydrogenase kinase-1 (PDK1), a key mitochondrial enzyme overexpressed in cancer cells, redirects glucose metabolism from oxidative phosphorylation toward aerobic glycolysis. In this study, we tested the hypothesis that targeting PDK1 is a rational approach to synergize with cetuximab through ROS overproduction. We found that combination of PDK1 knockdown or inhibition by dichloroacetic acid (DCA) with ASCT2 knockdown or with cetuximab treatment induced ROS overproduction and apoptosis in HNSCC cells, and this effect was independent of effective inhibition of EGFR downstream pathways but could be lessened by N-acetyl cysteine, an anti-oxidative agent. In several cetuximab-resistant HNSCC xenograft models, DCA plus cetuximab induced marked tumor regression, whereas either agent alone failed to induce tumor regression. Our findings call for potentially novel clinical trials of combining cetuximab and DCA in patients with cetuximab-sensitive EGFR-overexpressing tumors and patients with cetuximab-resistant EGFR-overexpressing tumors.

Development of an In Situ Cancer Vaccine via Combinational Radiation and Bacterial-Membrane-Coated Nanoparticles.

Neoantigens induced by random mutations and specific to an individual's cancer are the most important tumor antigens recognized by T cells. Among immunologically "cold" tumors, limited recognition of tumor neoantigens results in the absence of a de novo antitumor immune response. These "cold" tumors present a clinical challenge as they are poorly responsive to most immunotherapies, including immune checkpoint inhibitors (ICIs). Radiation therapy (RT) can enhance immune recognition of "cold" tumors, resulting in a more diversified antitumor T-cell response, yet RT alone rarely results in a systemic antitumor immune response. Therefore, a multifunctional bacterial membrane-coated nanoparticle (BNP) composed of an immune activating PC7A/CpG polyplex core coated with bacterial membrane and imide groups to enhance antigen retrieval is developed. This BNP can capture cancer neoantigens following RT, enhance their uptake in dendritic cells (DCs), and facilitate their cross presentation to stimulate an antitumor T-cell response. In mice bearing syngeneic melanoma or neuroblastoma, treatment with BNP+RT results in activation of DCs and effector T cells, marked tumor regression, and tumor-specific antitumor immune memory. This BNP facilitates in situ immune recognition of a radiated tumor, enabling a novel personalized approach to cancer immunotherapy using off-the-shelf therapeutics.

Role of Stem Cells in Management of Experimentally Induced Hypothyroidism in Albino Rat

AbstractBackground: Hypothyroidism is a common condition with potentially devastating health consequences that spread world-wide. Stems cells (SCs) based therapy had showed promising benefits in treatment of many diseases on animal models and applied on some human clinical issues.Aim of Study: To investigate the effect of bone marrow mesenchymal stem cells (BM-MSCs) on the biochemical and histological structures of the thyroid gland after experimentally induced hypothyroidism in adult male albino rats.Material and Methods: Forty five adult male Sprague-Dawley albino rats were divided into 3 equal groups: Control group (Group I), Hypothyroidism induced group (Group II); received orally carbimazole 45mg/kg of rat/day for 5 days/week for 4 weeks and Induced hypothyroidism treated with BM-MSCs group (Group III); received oral dose of carbimazole 45mg/kg of rat/day for 5 days/week for 4weeks then received single intravenous injection of 1x106 cells of BM-MSCs. This group was subdivided into three equal subgroups according to the time of scarification; Subgroup III6: Scarified at the end of 6th week, Subgroup III8: Scarified at the end of 8th week and Subgroup III10: Scarified at the end of 10th week. At the end of the experiment, the hormones of thyroid function were measured and histological examination of the thyroid tissue were done. In addition to morphometric study and statistical analysis.Results: BM-MSCs injection to the induced hypothy-roidism rats showed that after 6 and 8 weeks, there were highly significant increase (p 0.05) of free T3, T4 and TSH compared to the induced hypothyroidism group. The histological examination of the induced hypothyroidism after 6 weeks of BM-MSCs injection showed variable degree of response. However, after 8 weeks of BM-MSCs injection, there was universal improvement of all examined fields that confirmed statistically by improvement of the heights of follicular cells, area percent of collagen fibers and area percent of colloid. On the other hand, after 10 weeks of BM-MSCs injection, there was marked regression in response to SCs treatment in all examined parameters.Conclusion: BM-MSCs injection almost restored the function and histological structures of the thyroid gland after 8 weeks. However, this improvement was declined after 10 weeks of BM-MSCs injection. So second dose is recommended.

Biocompatibility of new bioactive resin composite versus calcium silicate cements: an animal study

BackgroundThe purpose of this study was to compare the biocompatibility of three bioactive materials, namely ACTIVA bioactive restorative resin composite, iRoot BP plus and Mineral Trioxide Aggregate (MTA) Angelus-HP.MethodsSeventy-five Wistar male rats were subjected to subcutaneous implantation of four polyethylene tubes; one empty tube was used as control (Group 1), and the other tubes were filled with ACTIVA (Group 2), iRoot BP (Group 3) and MTA-HP (Group 4). Then, the rats were subdivided into 3 groups according to the sacrification time into one, two and 4 weeks (n = 25 rats). Tissue specimens were submitted to histopathological and immunohistochemical analysis of α-SMA and caspase 3.ResultsThe one-way Anova test revealed that ACTIVA group exhibited minimal inflammation in comparison to calcium silicate cements (iRoot BP and MTA-HP groups). iRoot BP group significantly revealed a more severe degree of chronic inflammation in comparison to other groups (P < 0.05). ACTIVA group showed marked regression of inflammation and fibrosis comparable to the control, while iRoot BP group revealed remarkable fibrosis and calcification, with less degrees in MTA-HP group (P < 0.05). Immunostaining of both α-SMA and caspase 3 revealed lower indexes in ACTIVA group consistent with the control (P < 0.05).ConclusionsACTIVA showed a higher degree of biocompatibility to subcutaneous tissues in comparison to both iRoot BP and MTA-HP cements in regard to decrease the intensity of inflammation, with subsequent fibrous connective tissue remodeling and better healing patterns.Clinical significancePreliminary data suggests that the application of ACTIVA in retrograde fillings.

Inhibiting BRAF Oncogene–Mediated Radioresistance Effectively Radiosensitizes BRAFV600E-Mutant Thyroid Cancer Cells by Constraining DNA Double-Strand Break Repair

Activating BRAF mutations, most commonly BRAFV600E, are a major oncogenic driver of many cancers. We explored whether BRAFV600E promotes radiation resistance and whether selectively targeting BRAFV600E with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAFV600E thyroid cancer cells to radiotherapy. Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAFV600E or vemurafenib in thyroid cancer cells. BRAFV600E thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAFV600E into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAFV600E mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAFV600E cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAFV600E cells following IR. BRAFV600E mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAFV600E mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAFV600E thyroid tumor xenografts. BRAFV600E mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAFV600E thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAFV600E-mutant thyroid cancer.

Rapid progression and recovery of massive gastric mucosal damage complicated by Henoch-Schönlein purpura in a patient with liver cirrhosis: A case report.

Endoscopic findings associated with gastric ischemia include friable gastric mucosa, irregular ulceration, petechiae and purpura. The etiology of gastric ischemia includes shock, sepsis, thrombosis, embolism and vasculitis. Henoch-Schönlein purpura (HSP) is a leukocytoclastic vasculitis that affects small vessels in the skin, kidney and gastrointestinal (GI) tract. Rapid progression of HSP manifested with acute gastric mucosal bleeding has been rarely reported. The present case study reports on a patient who had undergone endoscopic variceal ligation for esophageal varix bleeding and the gastric mucosa exhibited no evidence of HSP. After 9 days, reexamination using endoscopy to check for new-onset hematemesis revealed massive necrosis in the gastric mucosa, friable mucosal tissue and acute ischemic GI bleeding. The patient was treated with corticosteroid injections for 7 days, which caused a marked regression of the hemorrhagic gastric mucosa. The present case study reported on this patient with acute GI bleeding caused by HSP who was successfully treated with corticosteroids.