PHLPP2 is a member of the PHLPP family of phosphatases, known to suppress cell growth by inhibiting proliferation or promoting apoptosis. Oncogenic kinases Akt, S6K, and PKC, and pro-apoptotic kinase Mst1, have been recognized as functional targets of the PHLPP family. However, we observed that, in T-leukemia cells subjected to metabolic stress from glucose limitation, PHLPP2 specifically targets the energy-sensing AMP-activated protein kinase, pAMPK, rather than Akt or S6K. PHLPP2 dephosphorylates pAMPK in several other human cancer cells as well. PHLPP2 and pAMPK interact with each other, and the pleckstrin homology (PH) domain on PHLPP2 is required for their interaction, for dephosphorylating and inactivating AMPK, and for the apoptotic response of the leukemia cells to glucose limitation. Silencing PHLPP2 protein expression prolongs the survival of leukemia cells subjected to severe glucose limitation by promoting a switch to AMPK-mediated fatty acid oxidation for energy generation. Thus, this study reveals a novel role for PHLPP2 in suppressing a survival response mediated through AMPK signaling. Given the multiple ways in which PHLPP phosphatases act to oppose survival signaling in cancers and the pivotal role played by AMPK in redox homeostasis via glucose and fatty acid metabolism, the revelation that AMPK is a target of PHLPP2 could lead to better therapeutics directed both at cancer and at metabolic diseases.

Breast cancer is the second leading cause of cancer-related mortalities in women. Triple-negative is the most lethal type of breast cancer with poorest survival rates. Triple-negative breast cancers do not respond to conventional hormone receptor targeted therapies and thus inhibitors for other overactivated signaling pathways are being investigated. The Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) is an oncogenic transcription factor pathway expressed in tumor and stromal cell populations. Inhibitors of this pathway such as ruxolitinib are being studied for treatment of triple-negative breast cancer but their efficacy in patients is limited. A significant barrier to the success of these inhibitors in the clinic is the lack of information about mechanisms of resistance. Tumor-associated macrophages (TAMs) are important components of the immune-suppressive environment within the tumor and are associated with poor outcome. We found that JAK inhibition in TAMs leads to induction of tumor-promoting genes that mediate therapeutic resistance of the tumor to JAK inhibition. A subset of the activated tumor promoting genes are known transcriptional targets of the NF-κB transcription factor pathway. We hypothesize that JAK inhibition activates tumor-promoting pathways in TAMs through activation of the NF-κB pathway and targeting these tumor-promoting pathways in combination with JAK inhibition will be beneficial over single therapy. We aim to elucidate activation of NF-κB in TAMs, to identify the mechanism of the observed pro-tumor response. In addition, we aim to target epiregulin, an EGFR ligand which was observed to be one of the most significantly upregulated genes in TAMs treated with JAK inhibitor. We hypothesize that targeting effects of macrophage-derived epiregulin in combination with JAK inhibition will overcome macrophage-mediated therapeutic resistance. This work will provide useful information about novel pathways that can be targeted to combat the compensatory negative effects of TAMs on tumor-targeted therapies. The results will also pave the way for development of novel inhibitor combinations targeting the tumor microenvironment in addition to targeting the tumor, and ultimately benefit patients who do not respond to single therapies. Citation Format: Aditi S. Bapat, Emily Jesser, Chelsea Lassiter, Kaylee Schwertfeger. Role of NF-κB and epiregulin in tumor-associated macrophages promoting therapeutic resistance to JAK inhibition [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO017.

Interactions between tumor cells and the tumor microenvironment are critical for tumor growth, progression, and response to therapy. Effective targeting of oncogenic signaling pathways in tumors requires an understanding of how these therapies impact both tumor cells and cells within the tumor microenvironment. One such pathway is the janus kinase (JAK)/signal transducer and activator or transcription (STAT) pathway, which is activated in both breast cancer cells and in tumor associated macrophages. This study demonstrates that exposure of macrophages to JAK inhibitors leads to activation of NF-κB signaling, which results in increased expression of genes known to be associated with therapeutic resistance. Furthermore, inhibition of the NF-κB pathway improves the ability of ruxolitinib to reduce mammary tumor growth in vivo. Thus, the impact of the tumor microenvironment is an important consideration in studying breast cancer and understanding such mechanisms of resistance is critical to development of effective targeted therapies.