Inhibition of B-cell lymphoma 2 family proteins alters optical redox ratio, mitochondrial polarization, and cell energetics independent of cell state.

SIGNIFICANCE: The optical redox ratio (ORR) [autofluorescence intensity of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)/flavin adenine dinucleotide (FAD)] provides a label-free method to quantify cellular metabolism. However, it is unclear whether changes in the ORR with B-cell lymphoma 2 (Bcl-2) family protein inhibition are due to metabolic stress alone or compromised cell viability. AIM: Determine whether ABT-263 (navitoclax, Bcl-2 family inhibitor) changes the ORR due to changes in mitochondrial function that are independent of changes in cell viability. APPROACH: SW48 colon cancer cells were used to investigate changes in ORR, mitochondrial membrane potential, oxygen consumption rates, and cell state (cell growth, viability, proliferation, apoptosis, autophagy, and senescence) with ABT-263, TAK-228 [sapanisertib, mammalian target of rapamycin complex 1/2 (mTORC 1/2) inhibitor], and their combination at 24 h. RESULTS: Changes in the ORR with Bcl-2 inhibition are driven by increases in both NAD(P)H and FAD autofluorescence, corresponding with increased basal metabolic rate and increased mitochondrial polarization. ABT-263 treatment does not change cell viability or induce autophagy but does induce a senescent phenotype. The metabolic changes seen with ABT-263 treatment are mitigated by combination with mTORC1/2 inhibition. CONCLUSIONS: The ORR is sensitive to increases in mitochondrial polarization, energetic state, and cell senescence, which can change independently from cell viability.

Impact of baseline culture conditions of cancer organoids when determining therapeutic response and tumor heterogeneity.

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. These results were corroborated using ~ 1700 patient-derived cancer organoids (PDCOs) isolated from 19 patients. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies.

Human Colon Organoids and Other Laboratory Strategies to Enhance Patient Treatment Selection.

OPINION STATEMENT: Though many advancements in personalized medicine have been made, better methods are still needed to predict treatment benefit for patients with colorectal cancer. Patient-derived cancer organoids (PDCOs) are a major advance towards true personalization of treatment strategies. A growing body of literature is demonstrating the feasibility of PDCOs as an accurate and high-throughput preclinical tool for patient treatment selection. Many studies demonstrate that these cultures are readily generated and represent the tumors they were derived from phenotypically and based on their mutation profile. This includes maintenance of the driver muatations giving the cancer cells a selective growth advantage, and also heterogeneity, including molecular and metabolic heterogeneity. Additionally, PDCOs are now being utilized to develop patient biospecimen repositories, perform high to moderate-throughput drug screening, and to potentially predict treatment response for individual patients that are undergoing anti-cancer treatments. In order to develop PDCOs as a true clinical tool, further studies are required to determine the reproducibility and accuracy of these models to predict patient response.

Patient-Derived Cancer Organoid Cultures to Predict Sensitivity to Chemotherapy and Radiation.

PURPOSE: Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed. EXPERIMENTAL DESIGN: Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively. RESULTS: We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer. CONCLUSIONS: These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.

Targeted Therapy in Metastatic Colorectal Cancer: Current Standards and Novel Agents in Review.

PURPOSE OF REVIEW: Treatment options for patients with metastatic colorectal cancer continue to advance as the therapeutic implications of the molecular subtypes of this disease are becoming better understood. DNA sequencing and mismatch repair assessment are now standard of care analyses for patients with metastatic colorectal cancer Thi review describes important aspects of the biology of the clinically relevant molecular subtypes of colorectal cancer based on the current standard of care testing. In addition, the clinical treatment strategies available now and potentially in the future for these colorectal cancer subtypes are discussed. RECENT FINDINGS: Currently for metastatic colorectal cancer, standard of care molecular testing is done for mutations in exons 2, 3, and 4 of KRAS and NRAS, and BRAF V600E. Testing for mismatch repair (MMR) deficiency/microsatellite instability (MSI) status is also done. These aberrations are well known to change the clinical prognosis and guide patients' treatment strategies. Additionally, three new subtypes have emerged: PIK3CAmut, HER2 amplified, and NTRK fusions. With the addition of these emerging subtypes, tumor heterogeneity further validates the need to examine mCRC as a heterogeneous disease. Here we present recent exciting data from translational research and clinical trials exhibiting possible distinct treatment strategies for these different subtypes. SUMMARY: Altogether these data show promising treatment strategies for many of these well-known and emerging subtypes of mCRC. In addition, these also give better clinical prognostic and predictive information. We believe that as molecular testing expands PIK3CA mutation, HER2 amplification, and NTRK fusion molecular testing will be included in standard of care analyses. This incorporation of testing in clinical practice will generate further information regarding prognostic and therapeutic options for these and other CRC subtypes in the future.

Tissue-Specific Oncogenic Activity of KRASA146T.

KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor-induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. SIGNIFICANCE: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties.See related commentary by Hobbs and Der, p. 696.This article is highlighted in the In This Issue feature, p. 681.

MTORC1/2 Inhibition as a Therapeutic Strategy for PIK3CA Mutant Cancers.

PIK3CA mutations are common in clinical molecular profiling, yet an effective means to target these cancers has yet to be developed. MTORC1 inhibitors are often used off-label for patients with PIK3CA mutant cancers with only limited data to support this approach. Here we describe a cohort of patients treated with cancers possessing mutations activating the PI3K signaling cascade with minimal benefit to treatment with the MTORC1 inhibitor everolimus. Previously, we demonstrated that dual PI3K/mTOR inhibition could decrease proliferation, induce differentiation, and result in a treatment response in APC and PIK3CA mutant colorectal cancer. However, reactivation of AKT was identified, indicating that the majority of the benefit may be secondary to MTORC1/2 inhibition. TAK-228, an MTORC1/2 inhibitor, was compared with dual PI3K/mTOR inhibition using BEZ235 in murine colorectal cancer spheroids. A reduction in spheroid size was observed with TAK-228 and BEZ235 (-13% and -14%, respectively) compared with an increase of >200% in control (P < 0.001). These spheroids were resistant to MTORC1 inhibition. In transgenic mice possessing Pik3ca and Apc mutations, BEZ235 and TAK-228 resulted in a median reduction in colon tumor size of 19% and 20%, respectively, with control tumors having a median increase of 18% (P = 0.02 and 0.004, respectively). This response correlated with a decrease in the phosphorylation of 4EBP1 and RPS6. MTORC1/2 inhibition is sufficient to overcome resistance to everolimus and induce a treatment response in PIK3CA mutant colorectal cancers and deserves investigation in clinical trials and in future combination regimens.