Traversing industry and academia in biomedicine: the best of both worlds?

Careers in biomedicine can take many forms, and one common career decision facing scientists is whether to pursue jobs in academia or industry. In this Viewpoint article, four leading scientists who have spent time in both academia and industry provide their perspectives on both types of workplace, such as whether the environments are really as distinct as they are often perceived to be, as well as how academia-industry collaborations can be a driving force in biomedical research and translation.

The interferon-stimulated gene RIPK1 regulates cancer cell intrinsic and extrinsic resistance to immune checkpoint blockade.

Interferon-gamma (IFN-γ) has pleiotropic effects on cancer immune checkpoint blockade (ICB), including roles in ICB resistance. We analyzed gene expression in ICB-sensitive versus ICB-resistant tumor cells and identified a strong association between interferon-mediated resistance and expression of Ripk1, a regulator of tumor necrosis factor (TNF) superfamily receptors. Genetic interaction screening revealed that in cancer cells, RIPK1 diverted TNF signaling through NF-κB and away from its role in cell death. This promoted an immunosuppressive chemokine program by cancer cells, enhanced cancer cell survival, and decreased infiltration of T and NK cells expressing TNF superfamily ligands. Deletion of RIPK1 in cancer cells compromised chemokine secretion, decreased ARG1+ suppressive myeloid cells linked to ICB failure in mice and humans, and improved ICB response driven by CASP8-killing and dependent on T and NK cells. RIPK1-mediated resistance required its ubiquitin scaffolding but not kinase function. Thus, cancer cells co-opt RIPK1 to promote cell-intrinsic and cell-extrinsic resistance to immunotherapy.

Runx1 negatively regulates inflammatory cytokine production by neutrophils in response to Toll-like receptor signaling.

RUNX1 is frequently mutated in myeloid and lymphoid malignancies. It has been shown to negatively regulate Toll-like receptor 4 (TLR4) signaling through nuclear factor κB (NF-κB) in lung epithelial cells. Here we show that RUNX1 regulates TLR1/2 and TLR4 signaling and inflammatory cytokine production by neutrophils. Hematopoietic-specific RUNX1 loss increased the production of proinflammatory mediators, including tumor necrosis factor-α (TNF-α), by bone marrow neutrophils in response to TLR1/2 and TLR4 agonists. Hematopoietic RUNX1 loss also resulted in profound damage to the lung parenchyma following inhalation of the TLR4 ligand lipopolysaccharide (LPS). However, neutrophils with neutrophil-specific RUNX1 loss lacked the inflammatory phenotype caused by pan-hematopoietic RUNX1 loss, indicating that dysregulated TLR4 signaling is not due to loss of RUNX1 in neutrophils per se. Rather, single-cell RNA sequencing indicates the dysregulation originates in a neutrophil precursor. Enhanced inflammatory cytokine production by neutrophils following pan-hematopoietic RUNX1 loss correlated with increased degradation of the inhibitor of NF-κB signaling, and RUNX1-deficient neutrophils displayed broad transcriptional upregulation of many of the core components of the TLR4 signaling pathway. Hence, early, pan-hematopoietic RUNX1 loss de-represses an innate immune signaling transcriptional program that is maintained in terminally differentiated neutrophils, resulting in their hyperinflammatory state. We hypothesize that inflammatory cytokine production by neutrophils may contribute to leukemia associated with inherited RUNX1 mutations.

Unifying mechanism for different fibrotic diseases.

Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun-mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway.

Runx1 Deficiency Decreases Ribosome Biogenesis and Confers Stress Resistance to Hematopoietic Stem and Progenitor Cells.

The transcription factor RUNX1 is frequently mutated in myelodysplastic syndrome and leukemia. RUNX1 mutations can be early events, creating preleukemic stem cells that expand in the bone marrow. Here we show, counterintuitively, that Runx1-deficient hematopoietic stem and progenitor cells (HSPCs) have a slow growth, low biosynthetic, small cell phenotype and markedly reduced ribosome biogenesis (Ribi). The reduced Ribi involved decreased levels of rRNA and many mRNAs encoding ribosome proteins. Runx1 appears to directly regulate Ribi; Runx1 is enriched on the promoters of genes encoding ribosome proteins and binds the rDNA repeats. Runx1-deficient HSPCs have lower p53 levels, reduced apoptosis, an attenuated unfolded protein response, and accordingly are resistant to genotoxic and ER stress. The low biosynthetic activity and corresponding stress resistance provides a selective advantage to Runx1-deficient HSPCs, allowing them to expand in the bone marrow and outcompete normal HSPCs.

Loss of c-Cbl E3 ubiquitin ligase activity enhances the development of myeloid leukemia in FLT3-ITD mutant mice.

Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase (RTK) occur frequently in acute myeloid leukemia (AML), with the most common involving internal tandem duplication (ITD) within the juxtamembrane domain. Fms-like tyrosine kinase 3-ITD mutations result in a mislocalized and constitutively activated receptor, which aberrantly phosphorylates signal transducer and activator of transcription 5 (STAT5) and upregulates the expression of its target genes. c-Cbl is an E3 ubiquitin ligase that negatively regulates RTKs, including FLT3, but whether it can downregulate mislocalized FLT3-ITD remains to be resolved. To help clarify this, we combined a FLT3-ITD mutation with a loss-of-function mutation in the RING finger domain of c-Cbl that abolishes its E3 ligase activity. Mice transplanted with hematopoietic stem cells expressing both mutations rapidly develop myeloid leukemia, indicating strong cooperation between the two. Although the c-Cbl mutation was shown to cause hyperactivation of another RTK, c-Kit, it had no effect on enhancing FLT3-ITD protein levels or STAT5 activation. This indicates that c-Cbl does not downregulate FLT3-ITD and that the leukemia is driven by independent pathways involving FLT3-ITD's activation of STAT5 and mutant c-Cbl's activation of other RTKs, such as c-Kit. This study highlights the importance of c-Cbl's negative regulation of wild-type RTKs in suppressing FLT3-ITD-driven myeloid leukemia.

Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis.

Genomic studies have identified somatic alterations in the majority of myeloproliferative neoplasms (MPN) patients, including JAK2 mutations in the majority of MPN patients and CALR mutations in JAK2-negative MPN patients. However, the role of JAK-STAT pathway activation in different MPNs, and in patients without JAK2 mutations, has not been definitively delineated. We used expression profiling, single nucleotide polymorphism arrays, and mutational profiling to investigate a well-characterized cohort of MPN patients. MPN patients with homozygous JAK2V617F mutations were characterized by a distinctive transcriptional profile. Notably, a transcriptional signature consistent with activated JAK2 signaling is seen in all MPN patients regardless of clinical phenotype or mutational status. In addition, the activated JAK2 signature was present in patients with somatic CALR mutations. Conversely, we identified a gene expression signature of CALR mutations; this signature was significantly enriched in JAK2-mutant MPN patients consistent with a shared mechanism of transformation by JAK2 and CALR mutations. We also identified a transcriptional signature of TET2 mutations in MPN patent samples. Our data indicate that MPN patients, regardless of diagnosis or JAK2 mutational status, are characterized by a distinct gene expression signature with upregulation of JAK-STAT target genes, demonstrating the central importance of the JAK-STAT pathway in MPN pathogenesis.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells.

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

Preclinical evaluation of the WEE1 inhibitor MK-1775 as single-agent anticancer therapy.

Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775-induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC(50) of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1(S345), induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity.

Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors.

The high frequency of activating RAS or BRAF mutations in cancer provides strong rationale for targeting the mitogen-activated protein kinase (MAPK) pathway. Selective BRAF and MAP-ERK kinase (MEK) inhibitors have shown clinical efficacy in patients with melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the extracellular signal-regulated kinase (ERK) signaling pathway. Here, we describe the identification and characterization of SCH772984, a novel and selective inhibitor of ERK1/2 that displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency in tumor cells with mutations in BRAF, NRAS, or KRAS and induces tumor regressions in xenograft models at tolerated doses. Importantly, SCH772984 effectively inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor-resistant models as well as in tumor cells resistant to concurrent treatment with BRAF and MEK inhibitors. These data support the clinical development of ERK inhibitors for tumors refractory to MAPK inhibitors.

STAT5 is crucial to maintain leukemic stem cells in acute myelogenous leukemias induced by MOZ-TIF2.

MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2-induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2-transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5(-/-) MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5(+/+) MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option.

mTOR complex 1 plays critical roles in hematopoiesis and Pten-loss-evoked leukemogenesis.

The mechanistic target of rapamycin (mTOR) pathway serves as a key sensor of cellular-energetic state and functions to maintain tissue homeostasis. Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) function and is associated with leukemogenesis. However, the roles of the unique mTOR complexes (mTORCs) in hematopoiesis and leukemogenesis have not been adequately elucidated. We deleted the mTORC1 component, regulatory-associated protein of mTOR (Raptor), in mouse HSCs and its loss causes a nonlethal phenotype characterized by pancytopenia, splenomegaly, and the accumulation of monocytoid cells. Furthermore, Raptor is required for HSC regeneration, and plays largely nonredundant roles with rapamycin-insensitive companion of mTOR (Rictor) in these processes. Ablation of Raptor also significantly extends survival of mice in models of leukemogenesis evoked by Pten deficiency. These data delineate critical roles for mTORC1 in hematopoietic function and leukemogenesis and inform clinical strategies based on chronic mTORC1 inhibition.

Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL.

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

A functional role for the histone demethylase UTX in normal and malignant hematopoietic cells.

Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), an H3K27Me2/3 demethylase, has been implicated in development, self-renewal, and differentiation of various organs and embryonic stem cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral short hairpin RNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine bone marrow cells impairs their colony-forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1, and Scl in primary murine bone marrow cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1, and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.

Drug development and clinical trials--the path to an approved cancer drug.

Advances in our understanding of cancer biology have led to the discovery of a spectrum of new therapeutic targets. However, despite remarkable progress in the identification and characterization of novel mechanisms of the oncogenic process, the success rate for approval of oncology drugs remains low relative to other therapeutic areas. Innovative preclinical and clinical approaches, such as the use of advanced genomic technologies, as well as branched adaptive clinical trial designs, have the potential to accelerate the development and approval of highly effective oncology drugs, along with a matching diagnostic test to identify those patients most likely to benefit from the new treatment. To maximize the effectiveness of these new strategies, close collaboration between academic, industry, and regulatory agencies will be required. In this Review, we highlight new approaches in preclinical and clinical drug development that will help accelerate approval of drugs, and aim to provide more-effective treatments alongside companion diagnostic tests to ensure the right treatment is given to the right patient.

KIT with D816 mutations cooperates with CBFB-MYH11 for leukemogenesis in mice.

KIT mutations are the most common secondary mutations in inv(16) acute myeloid leukemia (AML) patients and are associated with poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11, the fusion gene generated by inv(16), for leukemogenesis. Here, we transduced wild-type and conditional Cbfb-MYH11 knockin (KI) mouse bone marrow (BM) cells with KIT D816V/Y mutations. KIT transduction caused massive BM Lin(-) cell death and fewer colonies in culture that were less severe in the KI cells. D816Y KIT but not wild-type KIT enhanced proliferation in Lin(-) cells and led to more mixed lineage colonies from transduced KI BM cells. Importantly, 60% and 80% of mice transplanted with KI BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, whereas no control mice died. Results from limiting dilution transplantations indicate higher frequencies of leukemia-initiating cells in the leukemia expressing mutated KIT. Signaling pathway analysis revealed that p44/42 MAPK and Stat3, but not AKT and Stat5, were strongly phosphorylated in the leukemia cells. Finally, leukemia cells carrying KIT D816 mutations were sensitive to the kinase inhibitor PKC412. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis.

AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias.

AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ∼40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.

Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q- syndrome.

Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q- syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome.

Differential niche and Wnt requirements during acute myeloid leukemia progression.

Hematopoietic stem cells (HSCs) engage in complex bidirectional signals with the hematopoietic microenvironment (HM), and there is emerging evidence that leukemia stem cells (LSCs) may use similar interactions. Using a syngeneic retroviral model of MLL-AF9 induced acute myeloid leukemia (AML), we have identified 2 different stages of leukemia progression, propagated by "pre-LSCs" and established leukemia (LSCs) and compared the homing properties of these distinctive entities to that of normal HSCs. The homing and microlocalization of pre-LSCs was most similar to long-term HSCs and was dependent on cell-intrinsic Wnt signaling. In contrast, the homing of established LSCs was most similar to that of committed myeloid progenitors and distinct from HSCs. Although osteoblast-derived Dickkopf-1, a potent Wnt inhibitor known to impair HSC function, dramatically impaired normal HSC localization within the bone marrow, it did not affect pre-LSCs, LSC homing, or AML development. Mechanistically, cell-intrinsic Wnt activation was observed in human and murine AML samples, explaining the independence of MLL-AF9 LSCs from niche-derived Wnt signals. These data identify differential engagement of HM associated with leukemic progression and identify an LSC niche that is physically distinct and independent of the constraints of Wnt signaling that apply to normal HSCs.