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Medulloblastoma (MB) is a pediatric malignant brain tumor composed of four different subgroups (WNT, SHH, Group 3, Group 4), each of which are a unique biological entity with distinct clinico-pathological, molecular, and prognostic characteristics. Although risk stratification of patients with MB based on molecular features may offer personalized therapies, conventional subgroup identification methods take too long and are unable to deliver subgroup information intraoperatively. This limitation prevents subgroup-specific adjustment of the extent or the aggressiveness of the tumor resection by the neurosurgeon. In this study, we investigated the potential of rapid tumor characterization with Picosecond infrared laser desorption mass spectrometry (PIRL-MS) for MB subgroup classification based on small molecule signatures. One hundred and thirteen ex vivo MB tumors from a local tissue bank were subjected to 10- to 15-second PIRL-MS data collection and principal component analysis with linear discriminant analysis (PCA-LDA). The MB subgroup model was established from 72 independent tumors; the remaining 41 de-identified unknown tumors were subjected to multiple, 10-second PIRL-MS samplings and real-time PCA-LDA analysis using the above model. The resultant 124 PIRL-MS spectra from each sampling event, after the application of a 95% PCA-LDA prediction probability threshold, yielded a 98.9% correct classification rate. Post-ablation histopathologic analysis suggested that intratumoral heterogeneity or sample damage prior to PIRL-MS sampling at the site of laser ablation was able to explain failed classifications. Therefore, upon translation, 10-seconds of PIRL-MS sampling is sufficient to allow personalized, subgroup-specific treatment of MB during surgery. Significance: This study demonstrates that laser-extracted lipids allow immediate grading of medulloblastoma tumors into prognostically important subgroups in 10 seconds, providing medulloblastoma pathology in an actionable manner during surgery.

TAK-243 (MLN7243) is a first-in-class inhibitor of the ubiquitin-activating enzyme (UBA1) that catalyzes the first step in the ubiquitylation cascade whereby proteins are tagged with mono- or poly-ubiquitin to induce their degradation or modify their functions. Based on its preclinical efficacy and tolerability, TAK-243 has entered phase 1 clinical trials in advanced malignancies. However, the determinants of sensitivity to TAK-243 remain largely unknown. Therefore, we conducted a positive-selection, genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with lethal TAK-243 concentrations to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout conferred resistance to TAK-243 (FDR = 0.0012). BEND3-targeting gRNAs were enriched up to 10,000-fold after selection with the drug. To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells and confirmed the resistance phenotype. In vivo, tumors of BEND3-knockout cells were resistant to TAK243 (20 mg/kg twice weekly) as opposed to control tumors that showed dramatic reductions in tumor growth rate. As assessed by immunoblotting, BEND3 knockout dampened TAK-243 effects on ubiquitylation, proteotoxic stress and DNA damage response. Mechanistically, BEND3 knockout upregulated the ABC efflux transporter breast cancer resistance protein (BCRP; ABCG2), and decreased intracellular levels of TAK-243. It also conferred partial cross-resistance to pevonedistat and TAK-981–related selective inhibitors of the NEDD8-activating enzyme (NAE) and the SUMO-activating enzyme (SAE), respectively, as well as known substrates of BCRP (mitoxantrone and doxorubicin). Finally, TAK-243 sensitivity strongly correlated with BCRP expression in a panel of 30 cancer cell lines of different origin, and chemical inhibition of BCRP but not P-gp sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity. Citation Format: Samir H. Barghout, Ahmed Aman, Zachary Blatman, Karen Arevalo, Geethu Thomas, Neil MacLean, Xiaoming Wang, Rose Hurren, Troy Ketela, Moustafa Abohawya, Taira Kiyota, Rima Al-Awar, Aaron D. Schimmer. BEND3 modulates sensitivity to the UBA1 inhibitor TAK-243 by regulating expression of the multidrug transporter BCRP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6321.

HER2+ breast cancer is currently treated with chemotherapy plus anti-HER2 inhibitors. Many patients do not respond or relapse with aggressive metastatic disease. Therefore, there is an urgent need for new therapeutics that can target HER2+ breast cancer and potentiate the effect of anti-HER2 inhibitors, in particular those that can target tumor-initiating cells (TIC). Here, we show that MMTV-Her2/Neu mammary tumor cells cultured as nonadherent spheres or as adherent monolayer cells select for stabilizing mutations in p53 that “immortalize” the cultures and that, after serial passages, sphere conditions maintain TICs, whereas monolayer cells gradually lose these tumorigenic cells. Using tumorsphere formation as surrogate for TICs, we screened p53-mutant Her2/Neu+ tumorsphere versus monolayer cells with a lentivirus short hairpin RNA kinome library. We identified kinases such as the mitogen-activated protein kinase and the TGFβR protein family, previously implicated in HER2+ breast cancer, as well as autophagy factor ATG1/ULK1 and the noncanonical IκB kinase (IKK), TANK-binding kinase 1 (TBK1), which have not been previously linked to HER2+ breast cancer. Knockdown of TBK1 or pharmacologic inhibition of TBK1 and the related protein, IKKϵ, suppressed growth of both mouse and human HER2+ breast cancer cells. TBK1/IKKϵ inhibition promoted cellular senescence by suppressing p65–NF-κB and inducing p16Ink4a. In addition, TBK1/IKKϵ inhibition cooperated with lapatinib, a HER2/EGFR1–targeted drug, to accelerate apoptosis and kill HER2+ breast cancer cells both in culture and in xenografts. Our results suggest that patients with HER2+ breast cancer may benefit from anti-TBK1/IKKϵ plus anti-HER2 combination therapies and establish conditions that can be used to screen for additional TIC-specific inhibitors of HER2+ breast cancer. Cancer Res; 74(7); 2119–30. ©2014 AACR.

The transcription factor B cell lymphoma 6 (BCL6) is required for the generation of an effective humoral immune response through the development and maintenance of germinal centers (GCs). The inhibition of the protein−protein interaction between BCL6 and its corepressors has been implicated as a therapeutic target for diffuse large B-cell lymphoma (DLBCL), a type of non-Hodgkin’s lymphoma (NHL). Using structure-based drug design, we initiated a program to identify novel BCL6 inhibitors. We identified a high micromolar virtual screening hit which was then optimized for potent biophysical binding and anti-proliferative cellular activity resulting in the identification of OICR-10268, a potent and selective Bcl6 inhibitor. Citation Format: Iain D. Watson, Methvin Isaac, Brian Wilson, Anh Chau, Justin Morin, Pandiaraju Subramanian, Ahmed Mamai, Babu Joseph, Michael Prakesch, David Uehling, Ayome Abibi, Richard Marcellus, Craig Strathdee, Ratheesh Subramaniam, Brigitte Theriault, Jeffrey Winston, Manuel Chan, Carly Griffin, Herman Cheung, Taira Kiyota, Elijus Undzys, Ahmed Aman, Gennady Poda, Doug Kuntz, Neil C. Pomroy, Gil G. Privé, Rima Al-awar. Discovery of OICR-10268: A potent and selective BCL6 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 7.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the accumulation of malignant myeloid cells that have arrested maturation. Most therapeutic regimens approved or under development are cytotoxics. An alternate, but less explored therapeutic approach, is to induce terminal differentiation of AML cells. Upon differentiation, AML cells cease to proliferate or die. Phosphatidylserine decarboxylase (PISD) is a mitochondrial enzyme that converts phosphatidylserine (PS) to phosphatidylethanolamine (PE). Here, we explored the effects of inhibiting PISD on AML growth, stemness and differentiation. Knockout of PISD by CRISPR reduced the growth and clonogenic growth of OCI-AML2 cells. The reported chemical PISD inhibitor, 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine (aka: MMV007285), reduced growth and viability of OCI-AML2 cells (IC50 = 4.741 μM) and TEX cells (IC50 = 4.868 μM). Using the 8227 primary AML cell culture model, we showed that inhibiting PISD induced cell death in the functionally defined stem cell fraction (CD34+CD38-). MMV007285 also preferentially inhibited the clonogenic growth of primary AML cells (n = 7) over normal hematopoietic cells (n= 3). Moreover, MMV007285 induced AML cell differentiation as evidenced by increased CD11b expression and staining for non-specific esterase. Using high-performance thin layer chromatography (HPTLC), we found that inhibition of PISD with MMV007285 increased intracellular PS. To determine whether increased PS was functionally important, OCI-AML2 cells were treated with PS, resulting in reduced growth and clonogenic growth. Furthermore, PS supplementation targeted AML progenitor cells as it decreased engraftment of TEX cells in mice. Mechanistically, inhibiting PISD induced differentiation and decreased stemness in AML by activating Toll-like receptor (TLR) signaling. Specifically, inhibiting PISD upregulated TLR4 and 8 expression and increased expression of cytokines downstream of TLR activation. We also showed that TLR activation was functionally important to induce AML differentiation. Finally, we evaluated the effects of PISD inhibition in AML mouse models. MMV007285 (300 mg/kg/5 of 7 days orally for 10 days) decreased the growth of OCI-AML2 cells in SCID mice. Moreover, MMV007285 (150 mg/kg/5 of 7 days orally for 5 weeks) impeded the leukemic engraftment of primary AML cell in NOD/SCID mice without toxicity. Using secondary transplants, we showed that MMV007285 also targeted the leukemic stem cells. Taken together, inhibition of PISD altered phospholipid metabolism, inhibited growth and stemness, and increased differentiation in AML cells. Our findings reveal a previously undescribed link between mitochondrial phospholipid metabolism and AML stemness and differentiation, highlighting a potential new therapeutic strategy for AML. Citation Format: Mingjing Xu, Ayesh Seneviratne, Val A. Fajardo, Geethu E. Thomas, G. Wei Xu, Rose Hurren, S. Kim, Neil MacLean, Xiaoming Wang, Marcela Gronda, Danny Jeyaraju, Yulia Jitkova, David Sharon, Ahmed Aman, Rima Al-awar, Steven Chan, Mark D. Minden, Paul LeBlanc, Aaron D. Schimmer. Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3003.

Glioblastoma multiforme (GBM), characterized by an aggressive clinical course, therapeutic resistance, and striking molecular heterogeneity, remains incurable. Recent evidence, from our group and others, indicates that the JAK2/STAT3 pathway is an important mediator of tumor cell survival, growth, and invasion in GBM. We investigated the efficacy of a novel JAK2 inhibitor, pacritinib, in brain tumor initiating cell (BTIC) lines to evaluate its potential use in the treatment of GBM patients. In a Phase III study of patients with myelofibrosis, pacritinib demonstrated manageable toxicity and clinically and statistically improved patient spleen volume and reported outcomes. Treatment with pacritinib resulted in on-target JAK2/STAT3 inhibition at 1-2μM and dramatically reduced BTIC proliferation, regardless of endogenous MGMT promoter methylation or EGFR, PTEN, and TP53 mutational status. Pacritinib in combination with temozolomide, the current standard of care agent for GBM, prolonged survival over either drug alone in orthotopically xenografted NOD-SCID mice. We tested the hypothesis that combinatorial targeting of the JAK2/STAT3 pathway and other oncogenic drivers would be effective in GBM. A large number of GBMs have EGFR alterations and, despite poor clinical translation to date, EGFR inhibition remains of therapeutic relevance. Interestingly, EGFR inhibition leads to activation of survival-signalling pathways such as STAT3, diminishing the effectiveness of EGFR inhibition. We find that concurrent inhibition of JAK2/STAT3 and EGFR signalling may be an effective, clinically relevant therapeutic strategy for GBM. We examined the in vitro actions of pacritinib on BTICs in combination with clinically relevant EGFR inhibitors (erlotinib, afatinib, lapatinib and AZD9291). Combinatorial treatment with pacritinib and EGFR inhibitors showed striking responses, with lowered IC50s and BTIC viability. The combinatorial actions of EGFR and STAT3 inhibition were particularly effective in BTIC lines with EGFR activating vIII and missense point mutations. On-target activity was demonstrated with reduced phospho-EGFR, phospho-STAT3 and effectors of both pathways. In vivo pharmacokinetic and pharmacodynamic studies demonstrated that pacritinib and the EGFR inhibitors afatinib and AZD9291 penetrate the brain and have on-target activity. Ongoing in vivo studies using orthotopic xenograft BTIC models will determine whether combinatorial inhibition of these two pathways will provide survival benefit. Further studies are aimed at investigating whether combinatorial inhibition of other pro-oncogenic pathways, using the BTIC model both in vitro and in vivo, may be effective strategies in GBM. Citation Format: Hema A. Luchman, Katharine V. Jensen, Ahmed Aman, Samuel Weiss. Combinatorial strategies for glioblastoma using brain tumor-initiating cells: targeting the JAK/STAT and EGFR pathways. [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 279.

Excessive signaling from the Wnt pathway is associated with numerous human cancers. Using a high throughput screen designed to detect inhibitors of Wnt/β-catenin signaling, we identified a series of acyl hydrazones that act downstream of the β-catenin destruction complex to inhibit both Wnt-induced and cancer-associated constitutive Wnt signaling via destabilization of β-catenin. We found that these acyl hydrazones bind iron in vitro and in intact cells and that chelating activity is required to abrogate Wnt signaling and block the growth of colorectal cancer cell lines with constitutive Wnt signaling. In addition, we found that multiple iron chelators, desferrioxamine, deferasirox, and ciclopirox olamine similarly blocked Wnt signaling and cell growth. Moreover, in patients with AML administered ciclopirox olamine, we observed decreased expression of the Wnt target gene AXIN2 in leukemic cells. The novel class of acyl hydrazones would thus be prime candidates for further development as chemotherapeutic agents. Taken together, our results reveal a critical requirement for iron in Wnt signaling and they show that iron chelation serves as an effective mechanism to inhibit Wnt signaling in humans. Cancer Res; 71(24); 7628–39. ©2011 AACR.

Over the past few decades, extensive mouse and human genetics have implicated deregulated E2f and Cdk activity in cell exit escape and tumorigenesis. In flies, activating E2f or Cdk causes temporary abnormal division, but both must be induced for long-term aberrant proliferation - a hallmark in benign tumor-prone lesions. Almost invariably, defects in the Rb-E2f or Cip/Kip Cdk inhibitor (CKI)-Cdk2/1 cell cycle regulatory pathways occur together in human cancer. Despite this, little is known in regards to whether dual activation is an obligate feature for tumor susceptibility at initiation in vivo. In human retinoblastoma, homozygous RB1 inactivation is sufficient to initiate the disease. However, mouse retinoblastoma requires inactivation of both Rb1 and one of its family members (p107 or p130). Like pRb, p107 and p130 are best known as E2f inhibitors. Unexpectedly, the additional loss of p107 does not elevate E2f targets follow Rb loss. Instead, Rb/p107-deficient cells induce the E3 ubiquitin ligase Skp2 and activate Cdk2 in vivo. Strikingly, Cdk2 activity tightly correlates with susceptibility to retinoblastoma in our mouse models. Compiling this data, tumorigenesis may have two distinct steps: (1) Rb loss initiates abnormal division, and (2) the additional loss of p107 or, as we have found, p27kip1, both of which lead to Cdk2 activation is required to engender cancer susceptibility. Together, E2f and Cdk dual axes activity is essential for retinoblastoma initiation. To test this notion pharmaceutically, we treated retinoblastoma-prone mice with a Cdk or E2f small molecule inhibitor during the birth period of suspected cancer cell-of-origin. Strikingly, one-week exposure to an inhibitor of either axes curtailed retinoblastoma in Rb/p107- null and Rb/p27-null retinas. These therapeutic successes were achieved without disrupting normal proliferation. Thus, E2f inhibitors, untested in vivo, or Cdk inhibitors, largely unproven as therapeutics, may be ideal chemopreventative agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2590. doi:1538-7445.AM2012-2590