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The RAS→RAF→MEK→ERK pathway is hyperactivated in the majority of human lung adenocarcinoma (LUAD). However, the initial activating mutations induce homeostatic feedback mechanisms that limit ERK activity. How ERK activation reaches the tumor-promoting levels that overcome the feedback and drive malignant progression is unclear. We show here that the lung lineage transcription factor NKX2-1 suppresses ERK activity. In human tissue samples and cell lines, xenografts, and genetic mouse models, NKX2-1 induces the ERK phosphatase DUSP6, which inactivates ERK. In tumor cells from late-stage LUAD with silenced NKX2-1, re-introduction of NKX2-1 induces DUSP6 and inhibits tumor growth and metastasis. We show that DUSP6 is necessary for NKX2-1-mediated inhibition of tumor progression in vivo and that DUSP6 expression is sufficient to inhibit RAS-driven LUAD. Our results indicate that NKX2-1 silencing, and thereby DUSP6 downregulation, is a mechanism by which early LUAD can unleash ERK hyperactivation for tumor progression.

The RAS→RAF→MEK→ERK pathway is hyperactivated in the majority of human lung adenocarcinoma (LUAD). However, the initial activating mutations induce homeostatic feedback mechanisms that limit ERK activity. How ERK activation reaches the tumor-promoting levels that overcome the feedback and drive malignant progression is unclear. We show here that the lung lineage transcription factor NKX2-1 suppresses ERK activity. In human tissue samples and cell lines, xenografts, and genetic mouse models, NKX2-1 induces the ERK phosphatase DUSP6, which inactivates ERK. In tumor cells from late-stage LUAD with silenced NKX2-1, re-introduction of NKX2-1 induces DUSP6 and inhibits tumor growth and metastasis. We show that DUSP6 is necessary for NKX2-1-mediated inhibition of tumor progression in vivo and that DUSP6 expression is sufficient to inhibit RAS-driven LUAD. Our results indicate that NKX2-1 silencing, and thereby DUSP6 downregulation, is a mechanism by which early LUAD can unleash ERK hyperactivation for tumor progression.

Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2-1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies., eLife digest When cells become cancerous they grow uncontrollably and spread into surrounding healthy tissue. As the cancer progresses different genes are switched on and off which can cause tumor cells to change their identity and transition into other types of cell. How closely tumor cells resemble the healthy tissue they came from can influence how well the cancer responds to treatment. Many lung cancers have an identity similar to normal lung cells. However, some turn off a gene that codes for a protein called NKX2-1, which leads to a type of cancer called invasive mucinous adenocarcinoma (or IMA for short). Cells from this type of cancer develop an identity similar to mucous cells that line the surface of the stomach. But it was unclear how IMA tumor cells that developed from a mutation in the BRAF gene are affected by this loss in NKX2-1, and how transitioning to a different cell type impacts their response to treatment. To investigate this, Zewdu et al. studied lung cells from patients with IMA tumors driven by a mutation in BRAF and cells from mice that have been genetically engineered to have a similar form of cancer. This revealed that the NKX2-1 protein is needed to initiate the formation of cancer cells but is not required for the growth of already established BRAF-driven tumors. Further experiments showed that removing the gene for NKX2-1 made these cancer cells less responsive to drugs known as BRAF/MEK inhibitors that are commonly used to treat cancer. These drugs caused the IMA cancer cells to change their identity and become another type of stomach cell. This identity change was found to depend on two signaling pathways which cells use to communicate. This study provides some explanation of how IMA lung cancers that lack the gene for NKX2-1 resist treatment with BRAF/MEK inhibitors. It also shows new relationships between key genes in these cancers and systems for cell communication. These findings could lead to better therapies for lung cancer, particularly for patients whose tumor cells are deficient in NKX2-1 and therefore require specialized treatment.

Cancer cells often undergo lineage switching during their natural progression and in response to therapy. Lung adenocarcinomas (LUADs) exhibit a variety of differentiation states accompanied by dysregulation of lineage-specific transcription factors such as NKX2-1. Loss of NKX2-1 in human and murine LUAD leads to invasive mucinous adenocarcinoma (IMA), a subtype of lung cancer that exhibits pulmonary to gastric transdifferentiation. Human IMAs harbor a distinct spectrum of mutationally activated driver oncogenes compared to LUAD overall, suggesting that the transdifferentiation induced by NKX2-1 loss plays a context-dependent role in LUAD progression. Using genetically engineered mouse models, we find that NKX2-1 is required for optimal BRAF V600E driven lung tumor initiation but is dispensable for growth of established lung tumors. NKX2-1-deficient, BRAF V600E driven tumors morphologically resemble human IMA, have high levels of ERK phosphorylation and exhibit a distinct response to treatment with combined BRAF/MEK inhibitors. Whereas NKX2-1-positive tumor cells enter quiescence when treated with BRAF/MEK inhibitors, residual NKX2-1-negative cells fail to exit the cell cycle in response to the same therapy. Additionally, BRAF/MEK inhibitors induce canonical WNT signaling in NKX2-1-negative lung tumor cells, which is accompanied by cell identity switching within the gastric lineage. Co-inhibition of MAPK and WNT pathways blocked elements of this lineage switch in vitro and interfered with cell cycle changes imposed by MAPK inhibition in vivo. Our data show that there is a complex and reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of LUAD identity and suggest that lineage switching induced by targeted therapies may confer new therapeutic vulnerabilities.

The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE-class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme-specific inactivation of Pbx1 (SKO) on a Pbx2- or Pbx3-deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub-lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8 days following sublethal irradiation displayed significant downregulation of multiple cytokine-encoding genes, including KitL/SCF, Cxcl12/SDF-1, IL-3, IL-4, GM-CSF/Csf2 IL-10, and Igf-1, compared with controls. KitL/SCF and Cxcl12/SDF-1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non-cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF-1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury.

Morphogenesis of mammalian facial processes requires coordination of cellular proliferation, migration, and apoptosis to develop intricate features. Cleft lip and/or palate (CL/P), the most frequent human craniofacial birth defect, can be caused by perturbation of any of these programs. Mutations of WNT, P63, and IRF6 yield CL/P in humans and mice; however, how these genes are regulated remains elusive. We generated mouse lines lacking Pbx genes in cephalic ectoderm and demonstrated that they exhibit fully penetrant CL/P and perturbed Wnt signalling. We also characterized a midfacial regulatory element that Pbx proteins bind in order to control the expression of Wnt9b-Wnt3, which in turn regulate p63. Altogether, we establish a Pbx-dependent Wnt-p63-Irf6 regulatory module in midfacial ectoderm that is conserved within mammals. Dysregulation of this network leads to localized suppression of midfacial apoptosis and CL/P. Ectopic Wnt ectodermal expression in Pbx mutants rescues the clefting, opening avenues for tissue repair.

The post-cranial axial skeleton consists of a metameric series of vertebral bodies and intervertebral discs, as well as adjoining ribs and sternum. Patterning of individual vertebrae and distinct regions of the vertebral column is accomplished by Polycomb and Hox proteins in the paraxial mesoderm, while their subsequent morphogenesis depends partially on Pax1/Pax9 in the sclerotome. In this study, we uncover that Pbx1/Pbx2 are co-expressed during successive stages of vertebral and rib development. Next, by exploiting a Pbx1/Pbx2 loss-of-function mouse, we show that decreasing Pbx2 dosage in the absence of Pbx1 affects axial development more severely than single loss of Pbx1. Pbx1/Pbx2 mutants exhibit a homogeneous vertebral column, with loss of vertebral identity, rudimentary ribs, and rostral hindlimb shifts. Of note, these axial defects do not arise from perturbed notochord function, as cellular proliferation, apoptosis, and expression of regulators of notochord signaling are normal in Pbx1/Pbx2 mutants. While the observed defects are consistent with loss of Pbx activity as a Hox-cofactor in the mesoderm, we additionally establish that axial skeletal patterning and hindlimb positioning are governed by Pbx1/Pbx2 through their genetic control of Polycomb and Hox expression and spatial distribution in the mesoderm, as well as of Pax1/Pax9 in the sclerotome.

The clustering of neurons sharing similar functional properties and connectivity is a common organizational feature of vertebrate nervous systems. Within motor networks, spinal motor neurons (MNs) segregate into longitudinally arrayed subtypes, establishing a central somatotopic map of peripheral target innervation. MN organization and connectivity relies on Hox transcription factors expressed along the rostrocaudal axis; however, the developmental mechanisms governing the orderly arrangement of MNs are largely unknown. We show that Pbx genes, which encode Hox cofactors, are essential for the segregation and clustering of neurons within motor columns. In the absence of Pbx1 and Pbx3 function, Hox-dependent programs are lost and the remaining MN subtypes are unclustered and disordered. Identification of Pbx gene targets revealed an unexpected and apparently Hox-independent role in defining molecular features of dorsally projecting medial motor column (MMC) neurons. These results indicate Pbx genes act in parallel genetic pathways to orchestrate neuronal subtype differentiation, connectivity, and organization.

© 2015. Published by The Company of Biologists Ltd. The architecture of an organ’s vascular bed subserves its physiological function and metabolic demands. However, the mechanisms underlying gross vascular patterning remain elusive. Using intravital dye labeling and 3D imaging, we discovered that systems-level vascular patterning in the kidney is dependent on the kinetics of vascular mural cell (VMC) differentiation. Conditional ablation of the TALE transcription factor Pbx1 in renal VMC progenitors in the mouse led to the premature upregulation of PDGFRβ, amaster initiator of VMC-blood vessel association. This precocious VMC differentiation resulted in nonproductive angiogenesis, abnormal renal arterial tree patterning and neonatal death consistent with kidney dysfunction. Notably, we establish that Pbx1 directly represses Pdgfrb, and demonstrate that decreased Pdgfrb dosage in conditional Pbx1 mutants substantially rescues vascular patterning defects and neonatal survival. These findings identify, for the first time, an in vivo transcriptional regulator of PDGFRβ, and reveal a previously unappreciated role for VMCs in systems-level vascular patterning.

Albright hereditary osteodystrophy is caused by heterozygous inactivating mutations in GNAS, a gene that encodes not only the alpha-chain of Gs (Galphas), but also NESP55 and XLalphas through use of alternative first exons. Patients with GNAS mutations on maternally inherited alleles are resistant to multiple hormones such as PTH, TSH, LH/FSH, GHRH, and glucagon, whose receptors are coupled to Gs. This variant of Albright hereditary osteodystrophy is termed pseudohypoparathyroidism type 1a and is due to presumed tissue-specific paternal imprinting of Galphas. Previous studies have shown that mice heterozygous for a targeted disruption of exon 2 of Gnas, the murine homolog of GNAS, showed unique phenotypes dependent on the parent of origin of the mutated allele. However, hormone resistance occurred only when the disrupted gene was maternally inherited. Because disruption of exon 2 is predicted to inactivate Galphas as well as NESP55 and XLalphas, we created transgenic mice with disruption of exon 1 to investigate the effects of isolated loss of Galphas. Heterozygous mice that inherited the disruption maternally (-m/+) exhibited PTH and TSH resistance, whereas those with paternal inheritance (+/-p) had normal hormone responsiveness. Heterozygous mice were shorter and, when the disrupted allele was inherited maternally, weighed more than wild-type littermates. Galphas protein and mRNA expression was consistent with paternal imprinting in the renal cortex and thyroid, but there was no imprinting in renal medulla, heart, or adipose. These findings confirm the tissue-specific paternal imprinting of GNAS and demonstrate that Galphas deficiency alone is sufficient to account for the hormone resistance of pseudohypoparathyroidism type 1a.

Abstract 2989 Sequential drug combination is a rational approach to maximize tumor killing and minimize side effects in cancer therapy. However, this is rarely achieved because the mechanism of drug action is often incompletely understood and the cell cycle specificity of individual drugs unknown. Dysregulation of cyclin-dependent kinase (CDK)4 and CDK6 is common in human cancer and precedes unrestrained proliferation of tumor cells in multiple myeloma (MM) patients, especially during refractory relapse. This highlights the critical importance of targeting CDK4/CDK6 in MM. We have now developed, for the first time, a novel therapeutic strategy to selectively inhibit CDK4/CDK6 in sequential combination with clinically relevant cytotoxic drugs for maximal tumor killing at reduced doses in MM. CDK4 and CDK6 promote reentry and progression of the cell cycle through G1. PD 0332991, the only known CDK4/CDK6-specific inhibitor, is potent, reversible and bioavailable. We showed that inhibition of CDK4/CDK6 with PD 0332991 induces early G1 arrest and upon release from the G1 block, synchronous progression to S phase and G2/M with exceptional precision and efficiency in MM cells in vitro and in animal models. This provides a unique means to determine the cell cycle targeting specificity of individual compounds for optimal combination. Simultaneous analysis of BrdU pulse-labeling (30 minutes) and DNA content per cell reveals that bortezomib, a reversible proteasome inhibitor; carfilzomib (PR-171), an irreversible selective inhibitor of the proteasom; and its oral analog ONX-0912 (PR-047) all preferentially target MM cells synchronized into S phase over those arrested in G1, but not cells in G2/M. On this basis, killing of myeloma cells by proteasome inhibitors is markedly enhanced in prolonged G1 arrest induced by PD 0332991 and further augmented during synchronous entry into and progression through S phase upon release from the G1 block, in vitro and in vivo in the native bone marrow niches. Induction of early G1 arrest by PD 0332991 requires Rb, the substrate of CDK4 and CDK6, but not p53. Importantly, the increase in carfilzomib, ONX-0912 or bortezomib mediated killing after S phase synchronization significantly surpasses the enrichment of S phase cells. It is in fact proportional to the time of prior G1 arrest. Kinetics analyses of global gene expression patterns, specific RNA and protein levels and functional shRNA interference show that prolonging early G1 arrest leads to time-dependent uncoupling of gene expression from the cell cycle. PD 0332991 withdraw rapidly restores the CDK4 and CDK6 catalytic activity and scheduled expression of cell cycle genes, hence synchronous progression to S phase and mitosis. This includes upregulation of cyclin A synthesis and Skp2 mediated ubiquitin-proteasome degradation of p27 for S phase entry, mini chromosome maintenance(MCM)s and thymidine kinase for DNA replication, and genes critical for G2/M checkpoint control and mitosis. However, it fails to fully reverse the metabolic impairment (altered glucose, nucleotide and ATP metabolism) induced in prolonged early G1 arrest. This culminates in the loss of IRF-4 required for myeloma survival and selective gain of pro-apoptotic Bim function in G1 arrest and Noxa in S phase in synergy with carfilzomib and bortezomib, which lowers the threshold for activation of the intrinsic apoptosis pathway. Selective inhibition of CDK4/CDK6 in sequential combination therapy thus not only halts tumor cell proliferation but also potently induces synergistic tumor killing. This sequential combination therapy has been implemented in a multi-center phase 1/2 clinical trial targeting CDK4/6 with PD 0332991 in combination with bortezomib and dexamethasone in relapsed refractory MM. Phase 1 data indicate that PD 0332991 is well tolerated, and directly and completely inhibits CDK4/CDK6 and the cell cycle in tumor cells in MM patients with promising clinical efficacy. Evidence from phase 2 trials of carfilzomib indicates that it is also well tolerated. The peripheral neuropathy commonly observed with bortezomib appears to be less severe and less frequent. Selective combination with carfilzomib or the oral agent ONX-0912 thus represents a promising alternative to refine targeting CDK4/6 in sequential combination therapy for multiple myeloma and potentially other cancers. Disclosures: Off Label Use: PD 0332991 is a cell cycle CDK4/CDK6 inhibitor Carfilzomib is a proteasome inhibitor. Kirk:Onyx: Employment, Equity Ownership. Randolph:Pfizer: Employment, Equity Ownership. Niesvizky:Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau.

Abstract 2854 Poster Board II-830 Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Progression in multiple myeloma (MM) stems from both loss of apoptotic control in the bone marrow (BM) microenvironment and dysregulation of the cyclindependent kinases (CDK)4 and CDK6, which precedes uncontrolled proliferation of myeloma cells in vivo in particular during relapse. This reinforces the critical importance of targeting CDK4/CDK6 in MM. Through selective and reversible inhibition of CDK4/CDK6 with PD 0332991, the only known CDK4/6-specific inhibitor, we have recently developed a novel strategy to sensitize primary myeloma cells for cytotoxic killing by diverse cytotoxic drugs. These include carfilzomib (PR-171), an irreversible selective inhibitor of the chymotrypsin-like activity of the proteasome, and PR-047, an orally bioavailable analog of carfilzomib. We showed that induction of prolonged early G1 arrest following inhibition of CDK4/CDK6 markedly enhances cytotoxic killing of primary BM myeloma cells by either carfilzomib or PR-047 despite protection by BM stromal cells. The enhancement of cytotoxic killing is further augmented during synchronous S phase entry upon removal of PD 0332991 subsequent to induction of prolonged G1 arrest in myeloma cell lines. In both cases, the enhancement in carfilzomib (or PR-047) mediated killing is not associated with cell cycle regulation of the proteasome activity. It is caspase-dependent, requiring only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 30 nM. This killing is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9. Further, it is apparently initiated by cell cycle-dependent expression of the pro-apoptotic BH3-only proteins, which neutralize the anti-apoptotic Bcl-2 family proteins upstream of mitochondrial depolarization. Bim is upregulated during early G1 arrest to neutralize the anti-apoptotic MCL-1 and Bcl-2. By contrast, Noxa is silenced in G1 but dramatically upregulated in S phase, in particular when combined with carfilzomib. Importantly, targeting CDK4/CDK6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/CDK6 in combination with carfilzomib (or PR-047), therefore, not only halts tumor cell proliferation but also potently induces synergistic killing that is likely to profoundly inhibit cell cycle reentry and self-renewal in MM. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as indicated by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase 2 trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with the proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfilzomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies. Disclosures: Off Label Use: PD 0332991 is going to be used as a CDK4/6-specific inhibitor. Parlati:Proteolix, Inc.: Employment, Equity Ownership. Aujay:Proteolix, Inc.: Employment, Equity Ownership. Niesvizky:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Seattle Genetics, Inc: Research Funding; Proteolix: Research Funding, data monitoring committee.