CCNE2

APR-1051 is a highly selective and potentially best-in-class oral WEE1 inhibitor Phase 1 ACESOT-1051 clinical trial is evaluating APR-1051 as monotherapy treatment in patients with significant unmet medical need Dosing of the first patient in the ACESOT-1051 study represents a key advancement in Aprea’s clinical pipeline DOYLESTOWN, Pa., June 17, 2024 (GLOBE NEWSWIRE) -- Aprea Therapeutics, Inc. (Nasdaq: APRE) (“Aprea”, or the “Company”), a clinical-stage biopharmaceutical company focused on precision oncology through synthetic lethality, today announced that the first patient has been dosed in the ACESOT-1051 Phase 1 study evaluating daily oral WEE1 inhibitor APR-1051 as monotherapy in advanced solid tumor patients with unmet medical need. APR-1051 was discovered and preclinically evaluated by Aprea’s team of chemists and scientists. APR-1051 is a potent and highly selective small molecule designed to limit off-target toxicity that may provide good safety and tolerability and has shown a potentially favorable drug exposure in pre-clinical models. APR-1051 targets WEE1 kinase, an enzyme involved in the DNA damage response pathway. Based on preclinical studies, we believe APR-1051 may solve liabilities associated with other WEE1 inhibitors and is differentiated based on: 1) molecular structure; 2) selectivity for WEE1 versus off-target inhibition of the polo-like kinase, or PLK, family of kinases; 3) potentially improved pharmacokinetic (PK) properties; and 4) potential absence of QT prolongation at doses that significantly inhibit WEE1. No head-to-head studies with APR-1051 have been conducted. ACESOT-1051 is a focused biomarker-driven study with advanced/metastatic solid tumors harboring the following cancer-associated gene alterations: Amplification/overexpression of CCNE1 or CCNE2 regardless of tumor type, or Deleterious mutations in FBXW7 or PPP2R1A regardless of tumor type, or Colorectal cancer with KRAS-GLY12 and TP53 co-mutation, or Uterine serous carcinoma regardless of biomarker status “Dosing of the first patient in the ACESOT-1051 study is an important milestone in our APR-1051 development program and represents a key advancement of our clinical pipeline,” said Oren Gilad, Ph.D., President and Chief Executive Officer of Aprea. “Adding a second clinical program enriches our asset portfolio. We are initially evaluating single agent activity of APR-1051 to provide the basis for future rational combination treatments. We hope to confirm APR-1051’s safety pro this Phase 1 study and generate the necessary data that will help us understand how it can be best utilized to treat patients. We plan to provide a clinical update by year-end 2024 and generate preliminary efficacy data during 2025.” The first patient was enrolled at NEXT Oncology, San Antonio, Texas. Additional centers, including The University of Texas MD Anderson Cancer Center, are expected to participate. Anthony Tolcher M.D., Founder of Next Oncology commented, “NEXT Oncology is committed to exploring new treatment options for cancer patients and we are pleased to begin this important clinical trial. Cancers that over express Cyclin E (CCNE1 and CCNE2) represent a high unmet medical need, and patients with Cyclin E over expression have poor prognosis and no effective therapies. WEE1 kinase is a validated oncology target and we look forward to the results of this study.” ACESOT-1051 Study Design ACESOT-1051 (A Multi-Center Evaluation of WEE1 Inhibitor in Patients with Advanced Solid Tumors, APR-1051) is designed to assess the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of single-agent APR-1051 in advanced solid tumors harboring cancer-associated gene alterations. Oral APR-1051 will be administered once daily for 28-day cycles. The study consists of two parts: Part 1 is dose escalation and is expected to enroll up to 39 patients with advanced solid tumors. The first three dose levels will use accelerated titration followed by Bayesian Optimal Interval (BOIN) design for the remaining dose levels; Part 2 (up to 40 patients) is designed for dose optimization, with the goal of selecting the Recommended Phase 2 Dose (RP2D). The primary objectives of the study are to measure safety, dose-limiting toxicities (DLTs), maximum tolerated dose or maximum administered dose (MTD/MAD), and RP2D; secondary objectives are to evaluate pharmacokinetics, preliminary efficacy according to RECIST or PCWG3 criteria; pharmacodynamics is an exploratory objective. The University of Texas MD Anderson Cancer Center is the lead site, and the study will be performed at between 3 and 10 sites in the U.S. The ACESOT-1051 design was featured in a poster at the American Association of Cancer Research (AACR) annual meeting which took place in April 2024 in San Diego. A copy of the poster can be found here. For more information, refer to ClinicalTrials.gov NCT06260514. About Aprea Aprea Therapeutics, Inc. is a clinical-stage biopharmaceutical company headquartered in Doylestown, Pennsylvania, focused on precision oncology through synthetic lethality. The Company’s lead program is ATRN-119, a clinical-stage small molecule ATR inhibitor in development for solid tumor indications. APR-1051, an oral, small molecule WEE1 inhibitor, recently entered the clinic. For more information, please visit the company website at . The Company may use, and intends to use, its investor relations website at as a means of disclosing material nonpublic information and for complying with its disclosure obligations under Regulation FD. Forward-Looking Statement Certain information contained in this press release includes “forward-looking statements”, within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended related to our study analyses, clinical trials, regulatory submissions, and projected cash position. We may, in some cases use terms such as “future,” “predicts,” “believes,” “potential,” “continue,” “anticipates,” “estimates,” “expects,” “plans,” “intends,” “targeting,” “confidence,” “may,” “could,” “might,” “likely,” “will,” “should” or other words that convey uncertainty of the future events or outcomes to identify these forward-looking statements. Our forward-looking statements are based on current beliefs and expectations of our management team and on information currently available to management that involve risks, potential changes in circumstances, assumptions, and uncertainties. All statements contained in this press release other than statements of historical fact are forward-looking statements, including statements regarding our ability to develop, commercialize, and achieve market acceptance of our current and planned products and services, our research and development efforts, including timing considerations and other matters regarding our business strategies, use of capital, results of operations and financial position, and plans and objectives for future operations. Any or all of the forward-looking statements may turn out to be wrong or be affected by inaccurate assumptions we might make or by known or unknown risks and uncertainties. These forward-looking statements are subject to risks and uncertainties including, without limitation, risks related to the success, timing, and cost of our ongoing clinical trials and anticipated clinical trials for our current product candidates, including statements regarding the timing of initiation, pace of enrollment and completion of the trials (including our ability to fully fund our disclosed clinical trials, which assumes no material changes to our currently projected expenses), futility analyses, presentations at conferences and data reported in an abstract, and receipt of interim or preliminary results (including, without limitation, any preclinical results or data), which are not necessarily indicative of the final results of our ongoing clinical trials, our understanding of product candidates mechanisms of action and interpretation of preclinical and early clinical results from its clinical development programs, and the other risks, uncertainties, and other factors described under “Risk Factors,” “Management’s Discussion and Analysis of Financial Condition and Results of Operations” and elsewhere in the documents we the U.S. Securities and Exchange Commission. For all these reasons, actual results and developments could be materially different from those expressed in or implied by our forward-looking statements. You are cautioned not to place undue reliance on these forward-looking statements, which are made only as of the date of this press release. We undertake no obligation to update such forward-looking statements for any reason, except as required by law. Investor Contact: Mike Moyer LifeSci Advisors mmoyer@lifesciadvisors.com

Pre-clinical findings underscore the potential of APR-1051, a next-generation WEE1 kinase inhibitor, to be a well-tolerated and effective treatment for Cyclin E-overexpressing cancers IND for APR-1051 has been cleared; details on planned Phase 1 first in human trial (ACESOT-1051) presented ATRN-119, a novel macrocyclic ATR inhibitor, continues to appear safe and well tolerated with no Dose Limiting Toxicities observed in ongoing Phase 1/2a study; preliminary signs of clinical benefit reported; enrollment in the study continues April 10, 2024 -- Aprea Therapeutics, Inc. (Nasdaq: APRE) (“Aprea”, or the “Company”), a clinical-stage biopharmaceutical company focused on precision oncology through synthetic lethality, today released details about four poster presentations at the ongoing American Association of Cancer Research (AACR) Annual Meeting, taking place April 5 to 10, 2024 in San Diego, CA. The posters feature APR-1051, Aprea’s next-generation inhibitor of WEE1 kinase, as well as a clinical update on ATRN-119, its novel macrocyclic ATR inhibitor. The Company also presented a poster highlighting a new set of preclinical data in glioblastoma with a next-generation macrocyclic ATR inhibitor, ATRN-333. “The four poster presentations at this prestigious conference highlight our growing pipeline and commitment to help cancer patients in need,” said Dr. Oren Gilad, President and CEO of Aprea. “We are pleased to share the strong pre-clinical data and future clinical strategy for our promising next-generation WEE1 kinase inhibitor, APR-1051. We are also very excited to provide an encouraging update on the ongoing clinical study of our novel macrocyclic ATR inhibitor, ATRN-119.” Copies of the posters will be available on the Aprea corporate website here, at the conclusion of the AACR meeting. Lead Author and Presenter: Molly HansbargerAbstract Number: 7121 This poster summarizes the pre-clinical data of APR-1051 APR-1051 exhibits high potency for WEE1 inhibition in vitro Selectivity is key for success. APR-1051 shows low off-target inhibition of the PLK family of kinases. To measure the potential for off-target inhibition of the PLK family of enzymes, in vitro experiments were conducted to determine the IC50s of APR-1051 vs ZN-c3 (Zentalis Pharmaceuticals) The results showed significantly lower off-targeting of PLK1, PLK2 and PLK3 as indicated by higher IC50 values for APR-1051 compared to ZN-c3. IC50 of APR-1051 over IC50 of ZN-c3 • PLK1: > 150-fold • PLK2: > 50-fold • PLK3: > 600-fold • Off-targeting of PLK1 by other WEE1 inhibitors may compromise the efficacy of these drugs. • Off-targeting of the PLK family may increase the risk of producing PLKi-associated adverse effects. Cyclin E as a potential biomarker for APR-1051 treatment APR-1051 demonstrated effectiveness in suppressing the growth of Cyclin E-overexpressing breast and ovarian cancer cell lines. The dose and scheduling of APR-1051 that causes significant suppression of CCNE1-amplified high-grade serous ovarian cancer tumors in mice is well tolerated. Red blood cell and platelet counts remained within non-pathogenic ranges after a 28-day treatment period, consistent with proposed minimal off target PLK1 inhibition APR-1051 will potentially exhibit low cardiotoxicity. Inhibition WEE1 by APR-1051 occurs at an IC50 that is 200-fold lower on average than the IC50 of hERG potassium channel inhibition. Strong evidence for combination therapy APR-1051 was evaluated in combination with Aprea’s second-generation ATR inhibitors (ATRN-330 and ATRN-354) in xenografted tumors. The results showed higher anti-tumor activity for the combinations, compared with vehicle or monotherapy. APR-1051 received U.S. FDA clearance for a clinical trial, now with plans to dose the first patient in June 2024 Presenter: Nadeem Q. Mirza, M.D., MPHLead author: Timothy Yap, M.D.Abstract Number: CT196 This poster summarizes the strategy for the upcoming clinical trial of APR-1051 The aim of this first-in-human Phase 1 study (ACESOT-1051: A Multi-Center Evaluation of WEE1 Inhibitor in Patients with Advanced Solid Tumors, APR-1051) is to assess the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of single-agent APR-1051 in advanced solid tumors harboring cancer-associated gene alterations (NCT06260514) This biomarker-driven study will include patients with advanced/metastatic solid tumors harboring cancer-associated gene alterations, such as CCNE1 or CCNE2, FBXW7, PPP2R1A, or KRAS G12 Oral APR-1051 will be administered once daily for 28-day cycles. The study will consist of two parts. Part 1 will be dose escalation and is expected to enroll up to 39 patients with advanced solid tumors harboring cancer-associated gene alterations. In the dose escalation phase the first three dose levels will use accelerated titration followed by Bayesian Optimal Interval (BOIN) design for the remaining dose levels Part 2 (up to 40 patients) is designed for dose optimization, with the goal of selecting the Recommended Phase 2 Dose (RP2D) The primary objectives are to measure safety, dose-limiting toxicities (DLTs), maximum tolerated dose or maximum administered dose (MTD/MAD), RP2D; Secondary objectives are to evaluate pharmacokinetics, preliminary efficacy according to RECIST or PCWG3 criteria; Pharmacodynamics is an exploratory objective. Enrollment is anticipated to begin in Q2 2024 MD Anderson Cancer Center is the lead site, and the study will be performed at between 3 and 10 sites in the U.S Nadeem Mirza, MD, MPH, Senior Medical Advisor to Aprea commented, “Enrollment of patients continues in the dose escalation portion of our Phase 1/2a clinical trial evaluating ATRN-119 in patients with advanced solid tumors having mutations in defined DDR-related genes. We are now enrolling patients in the 550 mg cohort (Cohort 5). ATRN-119 continues to be safe and well tolerated, with no dose-limiting toxicities and no signs of significant hematological toxicity reported. We are encouraged by the preliminary signs of clinical benefit. Stable disease has been reported in two patients, one of which continues to be on treatment out to Day 188. Dose escalation will proceed throughout 2024.” First-in-human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors Presenter: Nadeem Q. Mirza, M.D., MPH Lead author: Fiona Simpkins, M.D. Abstract Number: CT195 This poster reports on the ongoing first-in-human Phase 1 study of ATRN-119 in patients with advanced solid tumors harboring specific DDR mutations (NCT04905914) As of March 12, 2024, 16 patients were enrolled in the first five cohorts of the dose escalation stage (50 mg/day, 100 mg/daily, 200 mg/daily, 350 mg/daily, and 550 mg/daily) ATRN-119 is being administered daily on a continuous schedule ATRN-119 has been found to be safe and well tolerated. No reported DLTs and no treatment-related Grade 4 or higher AEs have been reported. At doses up to 550 mg once daily, there have been no signs of hematological toxicity. Pharmacokinetic studies show ATRN-119 serum concentrations are entering the expected therapeutic range at the current highest dose level (550 mg). The Company currently has FDA clearance to evaluate doses up to 800mg, with a planned protocol amendment to add doses up to 1300 mg. Preliminary signs of clinical benefit have been observed. Two patients have achieved stable disease (SD) – one each in the 50 mg and 200 mg cohorts. The latter patient at 200 mg/day had SD at Days 55, 112, and 168, and continues to be on treatment as of Day 188 without significant adverse events reported. This patient is now receiving 350 mg daily, as per the trial protocol, and is tolerating treatment well. Convection-enhanced delivery of a novel ATR inhibitor synergizes with systemic lomustine for improved treatment of glioblastoma. Presenter: Teresa Lee, Ph.D. Lead Authors: Alexander Josowitz Ph.D., Teresa Lee Ph.D.Abstract Number: 7117 This poster describes a combination approach using a next-generation macrocyclic ATR inhibitor, ATRN-333, to sensitize glioblastoma (GBM) tumors to lomustine, an oral DNA alkylating agent. The DNA damage response and DNA repair mechanisms such as the ataxia telangiectasia and Rad3-related (ATR) pathway are key mediators of therapeutic responses in glioblastoma (GBM). Recent studies have shown that targeting DNA repair proteins alongside standard-of-care options is a promising anti-tumor strategy for this disease. To overcome difficulties associated with drug delivery to the brain, a convection-enhanced delivery (CED) system in conjunction with nanoparticle (NP) technology was used for direct intracranial administration of ATRN-333 to orthotopic GBM tumors. Both free and NP-encapsulated ATRN-333 showed high potency in inhibiting ATR function in cell-based assays. There was a clear synergistic effect between lomustine and ATRN-333 in GBM cell lines. ATRN-333 effectively sensitized both flank and intracranial tumors to lomustine in vivo. When administered via CED, ATRN-333 showed favorable intracranial retention and was well tolerated in mice when combined with lomustine. These results suggest that ATR inhibitor/lomustine combination therapy, used in conjunction with a CED platform, is a powerful avenue for GBM treatment. The results support further investigation and potential clinical implementation of ATRN-333 and other macrocyclic ATR inhibitors as chemosensitizers for glioblastoma. Aprea Therapeutics, Inc. is a clinical-stage biopharmaceutical company headquartered in Doylestown, Pennsylvania, focused on precision oncology through synthetic lethality. The Company’s lead program is ATRN-119, a clinical-stage small molecule ATR inhibitor in development for solid tumor indications. Aprea has completed all IND-enabling studies for its oral, small molecule WEE1 inhibitor, APR-1051, and recently received FDA clearance of its IND. For more information, please visit the company website at www.aprea.com. 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The roles and mechanisms of the m6A reader protein YTHDF1 in tumor biologyHepatocellular carcinoma (HCC) is the sixth most commonly diagnosed cancer and the third leading cause of cancer-related deaths worldwide. The development of HCC is influenced by both viral and non-viral factors. Non-alcoholic steatohepatitis (NASH) has emerged as a growing risk factor for HCC, as the prevalence of diabetes and metabolic syndrome increases. NASH is a progressive liver disease characterized by the accumulation of excessive triglycerides and is not caused by alcohol consumption. Although NASH is linked to the development of HCC, the molecular mechanisms underlying these diseases are not fully understood, which has hindered the development of effective treatments. Handicapping anti-PD1 therapyCurrently, there are no approved drugs or biological therapies specifically targeting NASH-related HCC (NASH-HCC). This deadly disease urgently requires new treatment methods. Immune checkpoint blockade (ICB) using monoclonal antibodies that target programmed cell death protein-1 (PD-1), programmed cell death-ligand 1 (PD-L1), or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has recently become a treatment option for HCC.  However, NASH-HCC's poor response to ICB treatment is due to NASH-related immune changes in the liver. Therefore, identifying potential therapeutic targets is critical to overcoming resistance to ICB treatment in NASH-HCC. YTHDF1, a m6A reader, is upregulated in human NASH-HCC tumors compared to peri-tumor tissues.N6-methyladenosine (m6A) is the most prevalent chemical modification in eukaryotic mRNA and is known to play a crucial role in post-transcriptional regulation. m6A modification regulates RNA fate by controlling RNA stability, processing, transport, localization, translation, and RNA-protein interactions. YTH domain-containing family protein 1 (YTHDF1) is a translation-promoting m6A reader that targets mRNAs in the cytoplasm and contributes to tumor progression by regulating cell proliferation, DNA damage, and immunity.  YTHDF1 deficiency in mice enhances antigen-specific CD8+ T cell-mediated anti-tumor responses in colorectal and gastric cancers, making it a potential therapeutic target for cancer immunotherapy. However, the role of YTHDF1 in the pathogenesis of HCC, particularly NASH-HCC, and its regulation of the tumor immune microenvironment are currently not well understood. A recent study published in Journal of Hepatology provides valuable insights into YTHDF1’s role in the progression of NASH-HCC. It is the first to demonstrate that tumor-intrinsic YTHDF1 is linked to the accumulation of myeloid-derived suppressor cells (MDSCs) in the NASH-HCC microenvironment. In transgenic mice with liver-specific YTHDF1 overexpression, YTHDF1 induces MDSC infiltration and accelerates spontaneous NASH-HCC.  Ythdf1 recruits MDSCs via IL-6 in NASH-HCC.The signaling cascade of YTHDF1 activates the EZH2-IL-6 signaling pathway to recruit MDSCs, resulting in T cell dysfunction and promoting tumor growth. Conversely, targeting YTHDF1 with lipid nanoparticle (LNP)-siRNA can significantly enhance the anti-PD-1 blockade effect in NASH-HCC mouse models, suggesting that YTHDF1 is a new target for enhancing the efficacy of immune checkpoint blockade therapy in NASH-HCC.  Targeting the accomplice: YTHDF1 According to the Synapse database, there are currently four patent applications targeting YTHDF1 for cancer therapy. Three of these applications are for the novel use of already existing drugs, while one is for a new formula: This is not the first time YTHDF1's role in cancer pathology has been elucidated. Last year, a study reported that YTHDF1 promotes breast cancer cell growth in both in vitro and in vivo environments and plays a crucial role in DNA damage repair and chemoresistance. And it seems that YTHDF1 modulates E2F8 mRNA stability and DNA damage repair in a METTL14-dependent manner. METTL14 is involved in DNA replication, DNA damage repair and chemoresistance.On the other hand, YTHDF1 knockdown has been shown to sensitize breast cancer cells to Adriamycin and Cisplatin, as well as Olaparib, a PARP inhibitor. During the experiments, researchers observed that the knockdown of YTHDF1 in MDA-MB-231 cells resulted in a reduction of S-phase fraction and an increased G1-phase fraction. The YTHDF1 knockdown also downregulated CDK2 and Cyclin E2, while upregulating P21, a CDK inhibitor involved in S-phase.   YTHDF1’s roles in other types of cancer have been revealed as well. A study published earlier this year has suggested that YTHDF1 may play an important role in shaping the immune microenvironment of colorectal cancer (CRC).  In their study, the team of researchers revealed that removing YTHDF1 from mouse CRC cells (both microsatellite stable (MSS) and microsatellite instable-high (MSI-H) varieties) results in an accumulation of myeloid-derived suppressor cells (MDSCs). This accumulation subsequently inhibits the infiltration of T cells in both mouse CRC models and humanized mouse models, demonstrating a deep interconnectedness. The investigation led to the identification of p65/Rela as a target of YTHDF1. The researchers observed that YTHDF1 encouraged the production of p65 protein, which in turn escalated the regulation of CXCL1. This process amplified the migration of MDSCs through the CXCL1-CXCR2 pathway, resulting in an increased number of MDSCs. These MDSCs then countered the function of CD8+ T cells within the tumor immune microenvironment (TIME). This intricate research, along with the others discussed here, suggest that the strategic targeting of YTHDF1, potentially using techniques such as CRISPR or VNPs-siYTHDF1, could enhance the efficacy of anti-PD1 therapy in a variety of cancers. References: 1.Wang L. et al., Targeting N6-methyladenosine reader YTHDF1 with siRNA boosts anti-tumor immunity in NASH-HCC by inhibiting EZH2-IL-6 axis 2, Journal of Hepatology, Available online 17 July 2023. 2.Sun, Y., Dong, D., Xia, Y. et al. YTHDF1 promotes breast cancer cell growth, DNA damage repair and chemoresistance. Cell Death Dis 13, 230 (2022). 3.Bao Y, Zhai J, Chen H, et al, Targeting m6A reader YTHDF1 augments antitumour immunity and boosts anti-PD-1 efficacy in colorectal cancer, Gut 2023;72:1497-1509.