Introduction Most cancer therapies fail due to the evolution of therapeutic resistance. Collateral sensitivity, wherein the evolution of resistance to one drug induces sensitivity to a second, has been extensively studied to tackle antibiotic resistance through sequential drug therapies. In cancers, similar evolutionary principles apply, and trade-offs can occur, whereby the cost of one resistance mechanism induces a population to be sensitive to an alternate therapy. Developing new methods to study treatment resistance through these evolutionary approaches is an important, yet understudied, need in the field. Material and methods To explore the evolutionary dynamics of treatment resistance, we extended an in vitro model system, initially in lung cancer cell lines, which enables the measurement of clonal dynamics using semi-random DNA barcode sequences introduced into cell lines via lentiviral transduction. One limitation of previous methods was the use of unrealistically small cell populations (105 – 106), which limits the genetic heterogeneity that is know to be pervasive in cancer. We utilised HYPERFlask technology to permit the growth of large cell populations (108), allowing us to explore drug treatments without the need for serial passaging that artificially reduces heterogeneity. Results and discussions By quantitative measurements of enrichment in molecular barcodes, we identified drug-specific patterns of selection under gefitinib and trametinib that were conserved between biological replicates. These quantitative analyses were indicative of distinct, pre-existing resistance within the treatment groups, including a small multi-drug resistant subclone. Further, we used high-throughput drug screening of the pre- and post-evolution replicates to survey sensitivities to new drugs. Through mathematical modelling, we extended these quantitative results to partially determine fitness landscapes and predict sequences of inhibitors wherein each sensitises the population to the next. Conclusion By combining evolutionary principles with mathematical modelling and quantitative measurements of evolution, we have derived a novel framework to identify collateral sensitivity. In comparison to previous approaches, this framework better captures the genetic heterogeneity that is intrinsic to many cancers, and which is known to drive drug resistance. We expect that this framework will now be used as treatment strategies for potential verification through in vivo and clinical studies.
Glucose uptake by tumors is stimulated by estradiol (E2) administration in estrogen receptor positive (ER+) breast cancer patients. Notably, this E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and, therefore, downstream metabolic regulators of E2 are expected to have utility as anti-breast cancer agents. Although the stimulation of glucose metabolism by E2 has been demonstrated, relatively little is known about the precise downstream effectors required for E2 to stimulate glucose metabolism in breast cancer. We have demonstrated that the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of the glycolytic flux, is elevated in breast cancer lymph node metastases and that exposure of ER+ human MCF-7 and T-47D breast cancer cells to E2 causes a rapid increase in 14C-glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, fructose-2,6-bisphosphate (F26BP). Importantly, we also found that selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor, PFK158, markedly reduces the E2-mediated increase in F26BP, 14C-glucose uptake and glycolysis. In the current study, we sought to determine if co-administration of PFK158, with ICI 182,780 (fulvestrant), would offer a greater anti-tumor effect. In unpublished results, we demonstrated that the combination of the anti-estrogen, fulvestrant, with PFK158 results in greater regressions of MCF-7 xenografts than either drug alone in athymic BALB/c mice. In addition to regulating glucose metabolism, PFKFB3 has been found to be a regulator of the cell cycle via cyclin dependent kinases. We postulated that the combination of PFK158 with the newly FDA-approved CDK4/6 inhibitor palbociclib may result in a synergistic decrease in tumor cell growth. We found that exposure of breast cancer cells to palbociclib and PFK158 causes a synergistic increase in cell cycle arrest and apoptotic cell death. In addition, we observed a synergistic decrease in phospho-retinoblastoma protein (Rb), a key downstream target of CDK4/6. Importantly, we found that the combination of PFK158 and palbociclib did not cause an increase in cell cycle arrest or apoptosis in normal human mammary epithelial cells. Taken together, these data indicate that PFKFB3 inhibitors such as PFK158 may have clinical utility for the treatment of ER+ breast cancers when combined with anti-estrogen agents and/or CDK4/6 inhibitors. Citation Format: Yoannis Imbert-Fernandez, Amy Clem, Brian Clem, Gilles Tapolsky, Sucheta Telang, Jason Chesney. Suppression of 6-Phosphofructo-2-Kinase (PFKFB3) for the treatment of breast cancer. [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 56.
Background: 6-phosphofructo-2-kinase (PFKFB3) is an enzyme that controls the intracellular concentration of fructose-2,6-bisphosphate which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a key enzyme of glycolysis. PFK-1 is tightly controlled by multiple metabolic feedback mechanisms and dictates the overall rate of glycolytic flux to lactate and the TCA cycle. In human cancers, several oncogenic proteins (e.g. HIF-1α, PTEN, and AKT) converge to increase the expression and activity of PFKFB3, leading to the high glycolytic rates typically observed in cancer cells. In addition to being a promising cancer metabolism target, PFKFB3 is required for the differentiation and tumor-promoting functions of the immunomodulatory Th17 cells and myeloid derived suppressor cells (MDSCs), which are attractive cellular targets to induce tumor immunity and potentially mediate intrinsic resistance to immune checkpoint inhibitors. PFK-158 is a potent selective small molecule inhibitor of PFKFB3 that displays broad anti-tumor activity causing significant growth inhibition in human and syngeneic preclinical models. As resistance mechanisms frequently activate pathways that result in up-regulation of glycolysis and PFKFB3, combination treatments of PFK-158 with cytotoxic and targeted agents have resulted in increased efficacy and tumor regression. Results: PFK-158 is a potent selective small molecule inhibitor of PFKFB3 that displays broad anti-tumor activity and causes significant growth inhibition in multiple human and syngeneic preclinical models. The tolerability and potential clinical benefit of PFK-158 are being investigated in advanced cancer patients with solid malignancies in a Phase 1 dose-escalation, multi-center clinical trial (clinicaltrials.gov # NCT02044861). The final cohort (650 mg/m2) has been open for enrollment and PFK-158 has been well tolerated to date. Secondary end-points have been incorporated to assess peripheral F2,6BP levels and immunosuppressive and effector cells populations. Of the 15 patients evaluable for response assessment at the end of two months of treatment, 6 patients have experienced a clinical benefit associated with PFK-158 administration, including a late stage pancreatic cancer patient that had a 75% reduction in her CA19-9 levels after 1 month, a renal cell carcinoma patient currently in month 9 and an adenocystic carcinoma patient in month 12. In addition, we examined the immunomodulatory effects of PFK-158 on Th17 cells and MDSCs in vitro, in B16 melanoma-bearing mice and in advanced cancer patients and found that PFK-158: (i) suppresses human Th17 cell and MDSC differentiation in vitro; (ii) decreases splenic and tumor-infiltrating Th17 cells, γδ T17 cells and MDSCs, and increases CD4+ and CD8+ T cells in the tumors of B16-F10 melanoma-bearing mice; and (iii) decreases peripheral blood Th17 cells, γδ T17 cells and MDSCs and increases activated effector CD4+ and CD8+ T cells in advanced cancer patients. Interestingly, we are discerning a correlation between the initial level of circulating Th17 cells and clinical responses to PFK-158. Conclusion: PFK-158 is the first-in-human and first-in-class PFKFB3 inhibitor that is currently under clinical development. To date, PFK-158 has been well tolerated and shows signs of clinical activity. In addition to controlling glycolysis, over expression of PFKFB3 in key immunesuppressive cells also leads to an immunomodulatory mechanism of action, suggesting that additional clinical benefit could result from combining PFK-158 with targeted agents as well as with immunotherapeutic agents. Note: This abstract was not presented at the conference. Citation Format: Sucheta Telang, Kavitha Yaddanapudi, Jaspreet Grewal, Rebecca Redman, Siqing Fu, Paula Pohlmann, Devalingam Mahalingam, Michael Kurman, Gilles Tapolsky, Jason Chesney.{Authors}. PFK-158 is a first-in-human inhibitor of PFKFB3 that selectively suppresses glucose metabolism of cancer cells and inhibits the immunosuppressive Th17 cells and MDSCs in advanced cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B90.
Estradiol (E2) administered to estrogen receptor positive (ER+) breast cancer patients stimulates glucose uptake by tumors. This E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and downstream metabolic regulators of E2 are expected to have utility as targets for the development of anti-breast cancer agents. While the stimulation of glucose metabolism by E2 has been demonstrated, relatively little is known about the precise downstream effectors required for E2 to stimulate glucose metabolism in breast cancer. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) control glycolytic flux via their product, fructose-2,6-bisphosphate (F26BP), which activates 6-phosphofructo-1-kinase (PFK-1). We recently demonstrated that PFKFB3 expression is elevated in breast cancer lymph node metastases and that exposure of human MCF-7 and T-47D breast cancer cells to E2 causes a rapid increase in 14 C-glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, F26BP. Importantly, we also found that selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor, PFK158, markedly reduces the E2-mediated increase in F26BP, 14 C-glucose uptake and glycolysis. In the current study, we sought to determine if co-administration of PFK158, with ICI 182,780 (fulvestrant), would offer a greater anti-tumor effect. In unpublished results, we demonstrated that the combination of the anti-estrogen, fulvestrant, with PFK158 results in greater regressions of MCF-7 xenografts than either drug alone in athymic BALB/C mice. In addition to regulating glucose metabolism, PFKFB3 has been found to be a regulator of the cell cycle via cyclin dependent kinases. We postulated that the combination of PFK158 with the newly FDA-approved CDK4/6 inhibitor palbociclib may result in a synergistic increase in apoptosis. We found that PFK158 markedly increased apoptosis caused by palbociclib in MCF-7 breast cancer cells but not in normal human mammary epithelial cells. Taken together, these data indicate that PFKFB3 inhibitors such as PFK158 may have clinical utility for the treatment of ER+ breast cancers when combined with anti-estrogen agents and/or CDK4/6 inhibitors. Citation Format: Yoannis Imbert-Fernandez, Brian Clem, Gilles Tapolsky, Jason Chesney. Regulation of 6-phosphofructo-2-kinase (PFKFB3) by estradiol and implications for the treatment of ER+ metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A84.
The optimal choice of cancer therapy depends upon analysis of the tumor genome for druggable molecular alterations. The spatial and temporal intratumor heterogeneity of cancers creates substantial challenges, as molecular profile depends on time and site of tumor tissue collection. To capture the entire molecular profile, multiple biopsies from primary and metastatic sites at different time points would be required, which is not feasible for ethical or economic reasons. Molecular analysis of circulating cell-free DNA offers a novel, minimally invasive method that can be performed at multiple time-points and plausibly better represents the prevailing molecular profile of the cancer. Molecular analysis of this cell-free DNA offers multiple clinically useful applications, such as identification of molecular targets for cancer therapy, monitoring of tumor molecular profile in real time, detection of emerging molecular aberrations associated with resistance to particular therapy, determination of cancer prognosis and diagnosis of cancer recurrence or progression.
