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Background: Pancreatic Cancer (PC) is one of the most aggressive cancer types, with less than 10% of patients surviving at 5-years. One hallmark of PC is the frequent mutation of the oncogene KRAS resulting in the constitutive activation of the phosphoinositide-3-kinase (PI3K) signaling pathway. The PI3K pathway is an intracellular signaling pathway that regulates cell growth, metabolism, and survival in response to extracellular signals. Given the importance of deregulated phosphoinositide (PI) signaling in PC, we hypothesize that genes coding for PI-metabolizing enzymes and effectors may have significant and unappreciated roles in PC. Methods: We generated a CRISPR knockout (KO) library that targets 1,534 PI-associated genes to discover novel essential genes in PC. Results: In a PANC-1 screen, we identified 106 essential genes across all timepoints, including 28 novel candidate genes and 78 previously identified essential genes. Among six validated candidate genes, we demonstrate that Integrin Subunit Alpha V (ITGAV) is a protein with clinical importance in PC. Treatment of PC cell lines with an ITGAV integrin antagonist as well as KO of ITGAV resulted in reduced cancer associated phenotypes in vitro, including reduced proliferation rate and compromised migration and invasion capacity. Ongoing studies will investigate perturbations in signaling pathways associated with ITGAV, and aim to elucidate roles for ITGAV in metastatic progression and therapy response. Conclusions: Overall, these findings indicate that ITGAV inhibition represents a putative strategy for the treatment of PC. Citation Format: Daniel K.C. Lee, Ryan Loke, Lydia To, Keyue Chen, Jonathan T.S. Chow, Leonardo Salmena. Phosphoinositide focused CRISPR screen reveals ITGAV as a critical gene in pancreatic cancer growth and invasion [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B024.

Signaling pathways that control vital features of leukemic stem cells including multipotency, self-renewal, clonal expansion and quiescence remain unclear. Emerging studies illustrate critical roles for lysosomes in hematopoietic and leukemic stem cell fate. By investigating consequences of INPP4B alterations in AML, we have discovered its role in driving leukemic ‘stemness’. We observed that INPP4B is highly expressed leukemic stem cell populations and Inpp4b-deficeint leukemias demonstrate increased disease latency, reduced leukemia initiating potential which is associated with a differentiated leukemic phenotype. Molecular analyses show that Inpp4b-deficient leukemias have compromised lysosomal gene expression, lysosomal content, and lysosomal activity. Our discovery of a novel pathway linking INPP4B, lysosomal biogenesis and leukemic stemness, provides a mechanism to explain the association of high INPP4B expression with poor AML prognosis, and highlights novel patient stratification strategies and LSC-specific leukemic therapies.Key PointsOur findings highlight a novel pathway linking INPP4B, lysosomal function and leukemic stemness that explains the prognostic role of INPP4B in AML.Our data reveal the utility of INPP4B as a biomarker of aggressive AML and provide a rationale to explore INPP4B and its associated function in lysosome biology as novel strategies to target LSC and AML

Acute myeloid leukemia (AML) is an aggressive clonal stem cell disorder characterized by invasion of bone marrow, impaired hematopoiesis and accumulation of functionally immature myeloblasts. Our previous studies demonstrated that the expression level of the lipid phosphatase, INPP4B, is associated with poor outcomes in AML. To gain an understanding of the role of Inpp4b in AML progression, we performed RNA sequencing on Inpp4b+/+ and Inpp4b−/− MLL-AF9 leukemias. Analysis of RNA sequencing data revealed that a disproportionately large number of lysosomal gene transcripts are decreased in Inpp4b−/− leukemias; these include cathepsins and lysosomal specific proteases responsible for proteolysis. This discovery provided evidence supporting a potential role for INPP4B in the lysosomal pathway. To validate these findings, immunofluorescence staining of the lysosomal protein LAMP1 was performed, which revealed a greater number of lysosomes in U2OS cells overexpressing INPP4B, and a decrease in lysosome number upon siRNA knockdown of INPP4B. Subsequently the DQ-BSA staining was used to assess INPP4B’s effect on lysosomal function. In both U2OS and the leukemia cells, we observed that INPP4B expression is positively associated with the proteolytic activity of lysosomes. Currently we are testing the role of INPP4B expression on sensitivity to lysosomal inhibitors; Lys05, Chloroquine, and Mefloquine. Overall, we aim to uncover how INPP4B expression in AML controls lysosomal mechanisms and biology and to test putative therapeutic strategies to exploit this pathway. Citation Format: Keyue Chen, Gizem E. Genc, John F. Woolley, Daniel K.C. Lee, Roberto J. Botelho, Leonardo Salmena. Investigating inositol polyphosphate-4-phosphatase, type II (INPP4B) signaling and role in acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P175.

Damaged DNA shows increased mobility, which can promote interactions with repair-conducive nuclear pore complexes (NPCs). This apparently random mobility is paradoxically abrogated upon disruption of microtubules or kinesins, factors that typically cooperate to mediate the directional movement of macromolecules. Here, we resolve this paradox by uncovering DNA damage-inducible intranuclear microtubule filaments (DIMs) that mobilize damaged DNA and promote repair. Upon DNA damage, relief of centromeric constraint induces DIMs that cooperate with the Rad9 DNA damage response mediator and Kar3 kinesin motor to capture DNA lesions, which then linearly move along dynamic DIMs. Decreasing and hyper-inducing DIMs respectively abrogates and hyper-activates repair. Accounting for DIM dynamics across cell populations by measuring directional changes of damaged DNA reveals that it exhibits increased non-linear directional behavior in nuclear space. Abrogation of DIM-dependent processes or repair-promoting factors decreases directional behavior. Thus, inducible and dynamic nuclear microtubule filaments directionally mobilize damaged DNA and promote repair., Following DNA damage, different processes come to action to aid repair. The authors here find that microtubule filaments within the cell nucleus capture and non-randomly mobilize damaged chromatin to mediate DNA repair.