ALBERT

Description: HER2 (ERBB2) amplification is a driving oncogenic event in breast cancer. Clinical trials have consistently shown the benefit of HER2 inhibitors (HER2i) in treating patients with both local and advanced HER2+ breast cancer. Despite this benefit, their efficacy as single agents is limited, unlike the robust responses to other receptor tyrosine kinase inhibitors like EGFR inhibitors in EGFR-mutant lung cancer. Interestingly, the lack of HER2i efficacy occurs despite sufficient intracellular signaling shutdown following HER2i treatment. Exploring possible intrinsic causes for this lack of response, we uncovered remarkably depressed levels of NOXA, an endogenous inhibitor of the antiapoptotic MCL-1, in HER2-amplified breast cancer. Upon investigation of the mechanism leading to low NOXA, we identified a micro-RNA encoded in an intron of HER2, termed miR-4728, that targets the mRNA of the Estrogen Receptor α (ESR1). Reduced ESR1 expression in turn prevents ERα-mediated transcription of NOXA, mitigating apoptosis following treatment with the HER2i lapatinib. Importantly, resistance can be overcome with pharmacological inhibition of MCL-1. More generally, while many cancers like EGFR-mutant lung cancer are driven by activated kinases that when drugged lead to robust monotherapeutic responses, we demonstrate that the efficacy of targeted therapies directed against oncogenes active through focal amplification may be mitigated by coamplified genes.

Description: Background: Venetoclax (VEN) monotherapy in patients (pts) with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) has been efficacious with a complete remission rate of 16% and also durable with median time to progression of 33.2 months (Roberts et al., 2019). Despite these responses, many pts eventually relapse. To investigate possible mechanisms of resistance to VEN monotherapy, we performed a genome-wide CRISPR screen to identify genes whose loss of expression leads to reduction in drug sensitivity. To determine whether similar genomic alterations occur in pts treated with VEN, we analyzed the DNA and RNA of pre- and post-therapy specimens from CLL pts enrolled in two VEN monotherapy trials (NCT01328626 and NCT01889186). Methods: CRISPR screen was performed in lymphoma SUDHL4 cell line using Brunello library. Peripheral blood or bone marrow specimens were collected pre-dose and after VEN discontinuation; CLL cells were enriched via CD19-positive (CD19+) selection using magnetic beads. DNA from 43 pts was analyzed by whole exome and two targeted sequencing panels. RNA-seq analysis was performed on paired pre- and post-therapy CD19+ samples from 24 pts. Results: The CRISPR screen identified genes whose loss of expression led to resistance to VEN. Several members of the pro-apoptotic machinery were among the strongest hits, including BAX, BCL2L11 (BIM) and PMAIP1 (NOXA). Outside of the apoptotic pathway, loss of ID3 or NFKBIA also led to decreased VEN sensitivity, suggesting multiple possible mechanisms of resistance. To compare these results to the potential mechanisms of resistance in pts treated with VEN, we sequenced the DNA from CD19+ B cells extracted before treatment initiation or upon treatment discontinuation. Of the 37 pts with disease progression, 15 acquired mutations in BCL2 at progression (N=14 CLL, N=1 Richters); 6 pts had a single mutation and 9 pts acquired 2-4 distinct BCL2 mutations. The prevalence of mutations increased with time on VEN therapy; mutations were observed in 12 pts progressing between 22 and 59 months and in only 3 pts progressing between 6 and 16 months. BCL2 mutations at G101V and D103 (E/Y/V) as previously described (Blombery et al., 2019; Tausch et al., 2019) were identified in 13/15 pts. Additionally, our analysis identified 1 patient with a mutation at F104L and 3 pts with mutations at A113G. These F104L and A113G mutations were previously identified in a preclinical NHL model of VEN resistance (Tahir et al., 2017) and in DLBLCL pts, respectively, but have not been previously reported in CLL pts. Two novel mutations in BCL2: a 4 amino acid insertion at position 110 and a substitution at V156(D) were acquired at CLL progression in 3 and 4 pts, respectively. Most BCL-2 mutations were subclonal (VAF 10%. In accordance with the CRISPR screen, we identified mutations in other pro-apoptotic genes including PMAIP1 (N=4), BAX (N=2) and BAD (N=1). In contrast to the acquired BCL2 mutations, mutations in these BCL-2 family members occurred with high allelic frequency in 4 of the 7 pts (27-35%). Interestingly, 4 pts had a mutation in BCL2 in addition to another member of the apoptotic machinery. In each case, allele frequency suggested that the mutations were found in different clones. Comparison of mRNA expression pre- and post-VEN treatment demonstrated increased expression at the time of progression of key apoptotic genes BCL2L1 (BCL-XL), MCL1 and BCL2A1, all of which are known resistance factors to VEN. Concomitant decreases in BCL2 and HRK expression, and to a lesser extent PMAIP1, were also observed. The changes in gene expression were found irrespective of the presence of BCL2 mutations. Conclusions: Preclinical investigation into potential resistance mechanisms indicated that reduction in several genes confer resistance to VEN. Some of these alterations were observed in clinical samples, including several distinct mutations in BCL2 and other BCL-2 family members.Our data suggest that BCL2 mutations are subclonal and further data are needed to evaluate their role in resistance to VEN monotherapy. Additional ongoing analyses of genomic data may identify genomic alterations in other biological pathways that potentially convey VEN resistance. Emergence of resistance is likely multifactorial with modulation of the apoptotic family members via both acquired mutations and changes in gene expression pattern. Disclosures Chyla: Abbvie, Inc: Employment, Other: Stock or options. Popovic:AbbVie: Employment, Other: Stock or options. Lu:AbbVie: Employment, Other: Stock or options. Dunbar:AbbVie: Employment, Other: Stock or options. Quarless:AbbVie: Employment, Other: Stock or options. Robinson:AbbVie: Employment, Other: Stock or options. Warder:AbbVie: Employment, Other: Stock or options. Jacobson:AbbVie: Employment, Other: Stock or options. Zhou:AbbVie: Employment, Other: Stock or options. Souers:AbbVie: Employment, Other: Stock or options. Waring:AbbVie: Employment, Other: Stock or options. Bhathena:AbbVie: Employment, Other: Stock or options. Leverson:AbbVie Inc: Employment, Other: Stock or options. Kim:AbbVie: Employment, Other: Stock or options.

Description: Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

Description: Background: Acute myeloid leukemia (AML) is characterized by the clonal expansion of immature myeloid cells. AML is treated primarily with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel therapies to achieve higher cure rates with more acceptable toxicities. Bcl-2 family proteins, together with TP53, are the central regulators of apoptosis. Overexpression of Bcl-2 protein is associated with leukemia progression and chemoresistance. We have observed elevated expression of Bcl-2 in AML and recently demonstrated that Bcl-2 inhibition by ABT-199 effectively induced apoptosis in AML (Pan, et.al., Cancer Discovery, 2014). However, resistance to ABT-199 was observed in cells expressing high levels of Mcl-1 or Bcl-xL. Moreover, a recent study showed heterogeneous but overlapping expression of Bcl-2, Mcl-1, and Bcl-xL proteins in 577 AML patient samples (Bogenberger, et. al., Leukemia, 2014). Although common in solid tumors, p53 mutations are relatively rare in AML. However, p53 functions are diminished by overexpression of MDM2 protein, an E3 ubiquitin ligase of p53 and an inhibitor of p53 transactivation. We previously reported MDM2 overexpression in 53% of primary AMLs (Kojima et al., Blood, 2005). Our group also demonstrated that p53 activation by Nutlins, the prototypical MDM2 inhibitors, induced apoptosis and growth inhibition in AML. Rationale: Since p53 activation by MDM2 inhibitors upregulates pro-apoptotic Bcl-2 proteins like NOXA, PUMA, and Bax, which counteract Mcl-1 and Bcl-xL, we hypothesized that the second-generation MDM2 inhibitor RG7388 could overcome AML resistance to Bcl-2-specific ABT-199, and that the combination could synergistically enhance apoptosis in AML. Results: We first demonstrated that RG7388 induced apoptosis exclusively in p53 wild type (wt) cells. RG7388 was essentially ineffective in p53 mutant or null AML cell lines such as HL-60, KG1 and THP1 (48h IC50s 〉 5 μM). Nonetheless, it showed high potency against p53 wt cell lines (48h IC50s: MOLM13 = 21.7 nM, MV-4-11 = 29.2 nM). Furthermore, stable knockdown of TP53 rendered the wt cell lines completely resistant to RG7388 (IC50s 〉 5 μM), confirming TP53-specificity. To study if RG7388 was able to overcome inherent resistance to ABT-199, we tested its efficacy on OCI-AML3 cells, which are inherently resistant to ABT-199, AraC and Idarubicin. As a single agent, RG7388 potently killed OCI-AML3 cells (48h IC50 = 148 nM). Importantly, RG7388 was ~20-fold more effective in OCI-AML3 cells than its predecessors Nutlin-3a and RG7112. We also examined the time- and dose-response of RG7388 in several genetically diverse AML cell lines (p53 wt) and found that 100 nM RG7388 was able to induce apoptosis and inhibit cell growth within 12 h. Next we studied whether RG7388 synergizes with ABT-199 to kill the refractory OCI-AML3 cells. A combination index of 0.35 (Chou-Talalay method) indicated a strong synergy between the two compounds. The combination exhibited higher activity in killing OCI-AML3 cells than either agent alone (48h IC50s: ABT-199 = 1680 nM, RG7388 = 148 nM, ABT+RG = 28 nM). Similar synergy was observed in additional AML cell lines and in primary samples. Next, we generated ABT-199 resistant cells by continuous exposure of initially sensitive AML cells to escalating concentrations of ABT-199. While 1000 nM ABT-199 had no effects on the viability of these cells, additional treatment with 30 nM RG7388 effectively killed them. This finding suggested that RG7388 was able to overcome acquired resistance to ABT-199. The mechanisms underlying this resensitization and its synergism with ABT-199 are under investigation using in vitro and in vivo model systems. Conclusions: The novel MDM2 inhibitor RG7388 induces growth arrest and apoptosis selectively in p53 wt AML cells. Importantly, the combination of RG7388 with ABT-199 synergistically induced apoptosis in AML cell lines and primary patient cells, and RG7388 was able to overcome inherent or acquired resistance to ABT-199. Since both Bcl-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical potential. We will report on ongoing experiments with primary AML cells in NSG mice to determine the potential of this combinatorial approach to eliminate AML stem cells. Disclosures Nichols: Roche: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership. Dangl:Roche: Employment, Patents & Royalties. Konopleva:Abbvie: Research Funding. Andreeff:Roche: Research Funding; Abbvie: Research Funding.