Triple negative breast cancer (TNBC) remains clinically challenging as patients have heterogeneous responses to current standard of care therapies. Chemotherapy sensitivity is a strong predictor of long-term outcomes for patients, and incomplete response of early stage disease to chemotherapy treatment is associated with a much higher risk of disease relapse and metastatic progression, often occurring within a short time from initial diagnosis. Therefore, treatment strategies that target chemotherapy-resistant TNBC and/or enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSC) have been proposed to represent a chemotherapy-resistant subpopulation within the tumor which are also responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. We have identified a novel multi-kinase inhibitor 108600 from a screen for inhibitors of this TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of the BCSC population. Treatment with 108600 induces G2M arrest and eventual apoptosis of TNBC cells in vitro and of TNBC xenografts in vivo, and overcomes chemotherapy (paclitaxel) resistance of triple negative patient-derived xenografts (PDX). Finally, treatment with 108600 and chemotherapy suppressed the growth of already established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.

No abstract

108600, a novel CK2/DYRK1/TNIK inhibitor, targets and inhibits cancer stem cells (CSC) in triple negative breast cancer (TNBC), inhibiting tumor growth and metastases in patient-derived xenograft models. CSC’s have been shown to promote immune evasion of several types of cancer. Specifically, over-expression of CK2 has been shown to promote intratumoral recruitment of myeloid derived suppressor cells. We investigated the effects of 108600 treatment on the immune microenvironment of triple negative breast cancer since targeting CSC’s could promote favorable anti-tumor immune responses and mechanistically contribute to tumor growth inhibition. Methods:C57BL/6 mice bearing murine triple negative E0771 tumors were generated by orthotopic injection and treated either with vehicle control or 108600 for 5 days. Tumor growth was assessed by daily caliper measurement All tumors were recovered at endpoint, and processed for RNA sequencing, flow cytometry or Western blot analysis. Results:108600 treatment significantly inhibited growth of E0771 tumors in vivo, demonstrating for the first time efficacy of 108600 against TNBC in immunocompetent models. 108600 treatment decreased intratumoral phosphorylation and expression of 108600 targets AKT1 (Ser 129) and Cyclin D1, which are substrates of CK2α and Dyrk1, respectively. To further explore nature of transcriptional and signaling pathways affected by 108600 treatment in vivo, RNA sequencing was performed. DGE and subsequent annotation analysis showed that various immune (GO:0006955, GO) and inflammatory (GO:0006954, GO) signaling pathways associated with Stat1 (mmu04062, KEGG) and IFNγ (mmu04060, KEGG) were down-regulated in 108600-treated E0771 tumors. GSEA (Gene set enrichment analysis) indicated that the regulatory T cell (Treg) population (GSE42021, GSE40685) was suppressed by 108600 treatment. To validate these findings, tumor infiltrating leukocytes (TIL) were isolated from vehicle or 108600-treated tumors and analyzed by flow cytometry. CD4, CD25, and FOXP3 expression was used to gate the Treg population. The Treg population was significantly suppressed by 108600 treatment, confirming the RNA sequencing results. 108600 also likely regulates expression of immunomodulatory molecules in TNBC tumor cells since 108600 treatment increased PD-L1 surface expression on E0771 cells in vitro. Conclusions:Our study supports 108600, an inhibitor that targets breast cancer stem cells, as a modulator of the immune microenvironment of TNBC. 108600 suppresses the Treg population among the TIL population and increases tumoral expression of PD-L1. Tregs have been associated with disease progression and metastases of TNBC. Further studies are ongoing to clarify the functional consequences of these changes in the setting of tumor growth inhibition. Synergies between 108600 and checkpoint inhibition are also under investigation and could lead to novel combination therapies for TNBC. Citation Format: Katsutoshi Sato, Stacey J. Baker, E. P. Reddy, Hanna Y. Irie. Novel cancer stem cell inhibitor 108600 modulates tumor immunomicroenvironment of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-08.

2944 Background: ON 01910.Na a novel benzyl styryl sulfone derivative is under clinical development in hematologic malignancies. It is a multi-kinase/PI3 kinase inhibitor that promotes G2/M arrest and selectively induces apoptosis in cancer cells. Leukemic cells exhibit significantly higher levels of sensitivity to ON 01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). Azacitidine (AzaC), is first line therapy for patients (pts) with higher-risk MDS and produces a response rate of 50%. Pts relapsed or refractory to hypomethylating based therapies have a poor prognosis and there are no accepted effective second line treatments, thus a need for new agents. Methods: A phase I/II study of ON 01910.Na is being conducted in pts with hematological malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks. A CBC is performed weekly and a bone marrow (BM) is performed at baseline and week 4, 8, and then q3 months thereafter. Pts with higher-risk disease had to have failed a hypomethylating agent. Results: Ten pts with MDS or AML relapsed/refractory to a hypomethylating agent have been treated with ON 01910.Na thus far (table 1). The study cohort comprised pts with a diagnosis (Dx) RAEB-2 (4 pts), RAEB-T (1 pt), and AML (5 pts) (median age of 75 years). Their cytogenetic profile included 1 pt with normal, 2 with intermediate (+8), and 7 pts with poor risk cytogenetics (monosomy 7 and/or complex). Patients were treated between 5 and 70 weeks. Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: Marrow CR (3), hematologic improvement (HI-P) (2); erythroid (1) platelet (1). An additional 2 pts had a >50% BM blast decrease from baseline but not to <5%. Thus, 5/10 (50%) demonstrate a bone marrow response. Survival of these pts was 7.3, 15.7, and 16.4 months; one patient remains on study 5+ months. Four of the five responders had MDS at the initiation of treatment: RAEB-2 (3), CMMoL (1), AML (1). Responders had monosomy 7 (2), trisomy 8 (1) and complex cytogenetics (2). One pt had an elimination of the MDS clone and the others had persistence of the abnormal karyotype throughout their treatment course. Five pts had SD without HI at 4 weeks, 2 pts progressed to AML. All 5 non-responders had AML; 4 with a proliferative course. These latter received only 2 (2) or 3 (3) cycles before succumbing to disease related infectious complications. Survival for these patients ranged from 1.3 – 2 months with a median duration on study of 42 days. The most frequent side effects grade2 2 for all pts included fatigue, anorexia, nausea, and dysuria in patients receiving extended duration infusions. One pt had a grade 3 urinary frequency. No hematologic toxicities occurred and no bone marrow toxicity or hypoplasia was noted. Pharmacokinetic studies are ongoing, data to date demonstrate no evidence of drug accumulation in patients who are treated repeatedly. Conclusion: ON 01910.Na appears to be safe and well tolerated in patients with refractory or relapsed MDS and AML. ON 01910.Na has biologic activity with reduction in BM blasts, eradication of the MDS clone and improvement in the peripheral blood counts in some pts. These effects are associated with increased survival albeit in limited numbers of pts treated thus far. Further study of ON 01910.Na is warranted to better define biologic activity, appropriate target populations and to define mechanism of action. Disclosures: Silverman: Onconova Therapeutics Inc: Research Funding, Research support of Clinical Trial. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership.

Methods: In the Austrian Biodatabase for CMML (ABCMML) real life data from CMML patients who are treated in different centers are collected centrally. Biologic assays are carried out in one laboratory in which in vitro cell cultures are performed. In this retrospective study data from the ABCMML were used to determine the frequency of myelomonocytic skewing, its correlation to hematologic and molecular parameters, and its prognostic impact in patients with CMML. 146 CMML patients were found in whom semisolid in vitro cell cultures from PBMNC have been performed between 1990 and 2018. The concentration of BFU-E and CFU-GM, respectively, was assessed as previously described (Geissler K et al, Blood 1996). For molecular characterization, we used NGS with amplicon-based target enrichment. Assuming that clones that are too small are unlikely to significantly impact hematopoiesis, only mutations with an allele burden of 20% or higher were considered as positive for analysis.Results: There was a high prevalence of myelomonocytic skewing as indicated by an inverse ratio of BFU-E/CFU-GM in CMML patients (120/146, 82%) whereas this phenomenon was rare in normal individuals (2/54, 4%). There was no difference in patients with and without myelomonocytic skewing with regard to age and male predominance. CMML patients with myelomonocytic skewing had higher WBC counts (17.